Antibacterial and antifungal peptides

ABSTRACT

This invention provides novel antimicrobial peptides and formulations thereof. The peptides and/or formulations are effective to kill or to inhibit the growth and/or proliferation of various bacteria, yeast, and fungi.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. Ser. No. 12/683,188, filed Jan.6, 2010 which claims benefit of and priority to U.S. Ser. No.61/142,830, filed Jan. 6, 2009, U.S. Ser. No. 61/151,445, filed Feb. 10,2009, U.S. Ser. No. 61/243,905, filed Sep. 18, 2009, and U.S. Ser. No.61/243,930, filed Sep. 18, 2009, all of which are incorporated herein byreference in their entirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

[Not Applicable]

FIELD OF THE INVENTION

The present invention relates to the fields of antibiotics andpharmacology. More particularly this invention pertains to theidentification of novel antimicrobial peptides (AMPs) that have activityagainst a number of bacteria and/or fungi.

BACKGROUND OF THE INVENTION

The development of antimicrobial agents led to a significant decrease inmorbidity and mortality from infectious diseases in this century. Thisaccomplishment was largely due to the widespread use of the majorclasses of antibiotics, such as the sulfonamides, penicillins,cephalosporins, aminoglycosides, and tetracyclines (see, e.g., Goodmanet al. (1995) The Pharmacological Basis of Therapeutics, MacmillanPublishing, New York). However, in recent years, the trend in reducinginfectious disease mortality has been threatened by the emergence ofresistant strains of microorganisms that are no longer susceptible tothe currently available antimicrobial agents.

With the rise of antibiotic-resistant pathogens and infectious diseases,the need for new antimicrobial agents is urgent (see, e.g., Cohen et al.(1992) Science 257: 1050-1055). For example, the incidence ofcommunity-acquired and nosocomially acquired infections due to thebacterium Staphylococcus aureus is rising (Lowy (1998) N. Engl. J. Med.339: 520-532.). From 1990 to 1992, this microorganism was the mostcommon cause of nosocomial pneumonias and surgical wound infections(Emori and Gaynes (1993) Clin. Microbiol. Rev. 23: 255-259). The overallgrowing crisis in antibiotic resistance and the rise in the incidence ofmethicillin-resistant S. aureus (MRSA) strains (Schwarz et al. (1981)Mol. Gen. Genet. 183:181-186; Sista et al. (2004) Anesthesiol. Clin. N.Am. 22: 405-435.) have emphasized the need for therapeutic alternativesto currently available antibiotics. Vancomycin remains the mainstay oftherapy against several resistant gram-positive pathogens. However,vancomycin is slowly bactericidal, and with the recent increase innosocomial infections caused by vancomycin-resistant enterococci and S.aureus (Centers for Disease Control and Prevention (2002) Morbid.Mortal. Wkly. Rep. 51: 565-567; Diekema et al. (2004) Clin. Infect. Dis.38: 78-85; Fujimura et al. (2004) J. Infect. Chemother., 10: 131-132),there is a growing need for antimicrobial agents with novel mechanismsof action to attack these resistant pathogens.

Biologically active peptides, such as antimicrobial peptides(hereinafter “AMPs”), are believed to be less likely to developresistance because the antimicrobial peptides show activity bymechanisms that are totally different from that of conventionalantibiotics.

Typically AMPs are low molecular weight peptides that exhibitantimicrobial activity. Naturally-occurring AMPs are part of the innateimmune response of plants, invertebrates and vertebrates. AMPs include,among others, cecropins (see, e.g., Hultmark et al. (1980) Eur. J.Biochem., 106: 7-16; Hultmark et al. (1982) Eur. J. Biochem., 127:207-217), apidaecins (see, e.g., Casteels et al. (1989) EMBO J. 8:2387-2391), magainins (see, e.g., Zasloff (1987) Proc. Natl. Acad. Sci.,USA, 84: 5449-5453; Zasloff et al. (1988) Proc. Natl. Acad. Sci., USA,85: 910-913), tachyplesins and analogues of tachyplesins such aspolyphemusins (see, e.g., Nakamura et al. (1988) J. Biol. Chem. 263:16709-16713; Miyata et al. (1989) J. Biochem., 106: 663-668), defensins(Lehrer et al. (1991) Cell 64: 229-230; Lehrer et al. (1993) Ann. Rev.Immunol., 11: 105-128; U.S. Pat. Nos. 4,705,777; No. 4,659,692;4,543,252), β-defensins (see, e.g., Selsted et al. (1993) J. Biol.Chem., 288: 6641-6648; Diamond et al. (1991) Proc. Natl. Acad. Sci.,USA, 88: 3952-3958), insect defensins (see, e.g., Lambert et al. (1989)Proc. Natl. Acad. Sci., USA, 88: 262-265; Matsuyama and Natori (1988) J.Biol. Chem., 263: 17112-17116), and protegrins (see, e.g., Kokryakov etal. (1993) FEBS 337: 231-236; Zhao et al. (1994) FEBS Lett. 346:285-288; Migorodskaya et al. (1993) FEBS 330: 339-342; Storici et al.(1993) Biochem. Biophys. Res. Commun., 196: 1363-1367; Zhao et al.(1994) FEBS Lett. 346: 285-288; Manzoni et al. (1996) FEBS Lett. 383:93-98; U.S. Pat. No. 5,464,823). The discovery of these new classes ofantimicrobial peptides offers hope that some might be developed intoagents that can be used against microorganisms of medicinal importance.

At least one antimicrobial peptide, daptomycin, a cycliclipodepsipeptide antibiotic, has been approved for the treatment ofcomplicated skin and skin structure infections caused by severalgram-positive bacteria. Its mode of action appears to be related to thedisruption of the membrane potential of the bacterium, which is causedby the favored oligomerization of daptomycin upon extracellular calciumbinding (Jeu and Fung (2004) Clin. Ther. 26: 1728-1757).

SUMMARY OF THE INVENTION

In various embodiments novel antimicrobial peptides (AMPs) are provided.The peptides are useful in a variety of contexts including, but notlimited to pharmaceuticals, and topical disinfectants.

Accordingly, certain embodiments provide an isolated antimicrobialpeptide having antimicrobial activity against at least one kind ofbacteria, fungus, or yeast, the antimicrobial peptide ranging in lengthup to about 80, about 70, or about 60 amino acids and comprising anamino acid sequence selected from group consisting ofGSVIKKRRKRMSKKKHRKMLRRTRVQRRKLGK (PF-S028, SEQ ID NO:1),NYRLVNAIFSKIFKKKFIKF (PF-C252, SEQ ID NO:2), YIQFHLNQQPRPKVKKIKIFL(PF-531, SEQ ID NO:3), GSVIKKRRKRMAKKKHRKLLKKTRIQRRRAGK (PF-527, SEQ IDNO:4), MRFGSLALVAYDSAIKHSWPRPSSVRRLRM (PF-672, SEQ ID NO:5),FESKILNASKELDKEKKVNTALSFNSHQDFAKAYQNGKI (PF-606, SEQ ID NO:6),KGKSLMPLLKQINQWGKLYL (PF-C239, SEQ ID NO:7), WSRVPGHSDTGWKVWHRW (PF-547,SEQ ID NO:8), MGIIAGIIKFIKGLIEKFTGK (PF-006, SEQ ID NO:9),ILNKKPKLPLWKLGKNYFRRFYVLPTFLA (PF-C287, SEQ ID NO:10), RESKLIAMADMIRRRI(PF-545, SEQ ID NO:11), LDPLEPRIAPPGDRSHQGAPACHRDPLRGRSARDAER (PF-0019,SEQ ID NO:12), MPVSKKRYMLSSAYATALGICYGQVATDEKESEITAIPDLLDYLSVEEYLL(PF-C163, SEQ ID NO:13), LSLATFAKIFMTRSNWSLKRFNRL (PF-278, SEQ IDNO:14), MIRIRSPTKKKLNRNSISDWKSNTSGRFFY (PF-283, SEQ ID NO:15),MKRRRCNWCGKLFYLEEKSKEAYCCKECRKKAKKVKK (PF-307, SEQ ID NO:16),VLPFPAIPLSRRRACVAAPRPRSRQRAS (PF-168, SEQ ID NO:17),KNKKQTDILEKVKEILDKKKKTKSVGQKLY (PF-538, SEQ ID NO:18), SLQSQLGPCLHDQRH(PF-448, SEQ ID NO:19), WKRLWPARILAGHSRRRMRWMVVWRYFAAT (PF-C021, SEQ IDNO:20), KFQGEFTNIGQSYIVSASHMSTSLNTGK (PF-583, SEQ ID NO:21),TKKIELKRFVDAFVKKSYENYILERELKKLIKAINEELPTK (PF-600, SEQ ID NO:22),KFSDQIDKGQDALKDKLGDL (PF-525, SEQ ID NO:23), LSEMERRRLRKRA (PF-529, SEQID NO:24), RRGCTERLRRMARRNAWDLYAEHFY (PF-148, SEQ ID NO:25),SKFKVLRKIIIKEYKGELMLSIQKQR (PF-530, SEQ ID NO:26), FELVDWLETNLGKILKSKSA(PF-522, SEQ ID NO:27), LVLRICTDLFTFIKWTIKQRKS (PF-497, SEQ ID NO:28),VYSFLYVLVIVRKLLSMKKRIERL (PF-499, SEQ ID NO:29), GIVLIGLKLIPLLANVLR(PF-322, SEQ ID NO:30), VMQSLYVKPPLILVTKLAQQN (PF-511, SEQ ID NO:31),SFMPEIQKNTIPTQMK (PF-512, SEQ ID NO:32),LGLTAGVAYAAQPTNQPTNQPTNQPTNQPTNQPTNQPRW (PF-520, SEQ ID NO:33),CGKLLEQKNFFLKTR (PF-521, SEQ ID NO:34), ASKQASKQASKQASKQASKQASRSLKNHLL(PF-523, SEQ ID NO:35), PDAPRTCYHKPILAALSRIVVTDR (PF-524, SEQ ID NO:36),NYAVVSHT (PF-209, SEQ ID NO:37), ILVLLALQVELDSKFQY (PF-C157, SEQ IDNO:38), YVNYNQSFNSGW (PF-C220, SEQ ID NO:39), andFQKPFTGEEVEDFQDDDEIPTII (PF-437, SEQ ID NO:40) and/or the inverse ofthese sequences. In certain embodiments the amino acid sequence of thepeptide consists of a sequence selected from group consisting ofGSVIKKRRKRMSKKKHRKMLRRTRVQRRKLGK (PF-5028, SEQ ID NO:1),NYRLVNAIFSKIFKKKFIKF (PF-C252, SEQ ID NO:2), YIQFHLNQQPRPKVKKIKIFL(PF-531, SEQ ID NO:3), GSVIKKRRKRMAKKKHRKLLKKTRIQRRRAGK (PF-527, SEQ IDNO:4), MRFGSLALVAYDSAIKHSWPRPSSVRRLRM (PF-672, SEQ ID NO:5),FESKILNASKELDKEKKVNTALSFNSHQDFAKAYQNGKI (PF-606, SEQ ID NO:6),KGKSLMPLLKQINQWGKLYL (PF-C239, SEQ ID NO:7), WSRVPGHSDTGWKVWHRW (PF-547,SEQ ID NO:8), MGIIAGIIKFIKGLIEKFTGK (PF-006, SEQ ID NO:9),ILNKKPKLPLWKLGKNYFRRFYVLPTFLA (PF-C287, SEQ ID NO:10), RESKLIAMADMIRRRI(PF-545, SEQ ID NO:11), LDPLEPRIAPPGDRSHQGAPACHRDPLRGRSARDAER (PF-0019,SEQ ID NO:12), MPVSKKRYMLSSAYATALGICYGQVATDEKESEITAIPDLLDYLSVEEYLL(PF-C163, SEQ ID NO:13), LSLATFAKIFMTRSNWSLKRFNRL (PF-278, SEQ IDNO:14), MIRIRSPTKKKLNRNSISDWKSNTSGRFFY (PF-283, SEQ ID NO:15),MKRRRCNWCGKLFYLEEKSKEAYCCKECRKKAKKVKK (PF-307, SEQ ID NO:16),VLPFPAIPLSRRRACVAAPRPRSRQRAS (PF-168, SEQ ID NO:17),KNKKQTDILEKVKEILDKKKKTKSVGQKLY (PF-538, SEQ ID NO:18), SLQSQLGPCLHDQRH(PF-448, SEQ ID NO:19), WKRLWPARILAGHSRRRMRWMVVWRYFAAT (PF-C021, SEQ IDNO:20), KFQGEFTNIGQSYIVSASHMSTSLNTGK (PF-583, SEQ ID NO:21),TKKIELKRFVDAFVKKSYENYILERELKKLIKAINEELPTK (PF-600, SEQ ID NO:22),KFSDQIDKGQDALKDKLGDL (PF-525, SEQ ID NO:23), LSEMERRRLRKRA (PF-529, SEQID NO:24), RRGCTERLRRMARRNAWDLYAEHFY (PF-148, SEQ ID NO:25),SKFKVLRKIIIKEYKGELMLSIQKQR (PF-530, SEQ ID NO:26), FELVDWLETNLGKILKSKSA(PF-522, SEQ ID NO:27), LVLRICTDLFTFIKWTIKQRKS (PF-497, SEQ ID NO:28),VYSFLYVLVIVRKLLSMKKRIERL (PF-499, SEQ ID NO:29), GIVLIGLKLIPLLANVLR(PF-322, SEQ ID NO:30), VMQSLYVKPPLILVTKLAQQN (PF-511, SEQ ID NO:31),SFMPEIQKNTIPTQMK (PF-512, SEQ ID NO:32),LGLTAGVAYAAQPTNQPTNQPTNQPTNQPTNQPTNQPRW (PF-520, SEQ ID NO:33),CGKLLEQKNFFLKTR (PF-521, SEQ ID NO:34), ASKQASKQASKQASKQASKQASRSLKNHLL(PF-523, SEQ ID NO:35), PDAPRTCYHKPILAALSRIVVTDR (PF-524, SEQ ID NO:36),NYAVVSHT (PF-209, SEQ ID NO:37), ILVLLALQVELDSKFQY (PF-C157, SEQ IDNO:38), YVNYNQSFNSGW (PF-C220, SEQ ID NO:39), andFQKPFTGEEVEDFQDDDEIPTII (PF-437, SEQ ID NO:40). In certain embodimentsthe peptide is effective to kill or inhibit the growth or proliferationof a yeast and/or fungus (e.g., A. niger, C. albicans, T. rubrum, M.furfur, etc.) where the amino acid sequence of the peptide comprises oneor more sequences selected from the group consisting of PF-148, PF-168,PF-448, PF-525, PF-527, PF-529, PF-531, PF-545, PF-672, PF-0019, PF-278,PF-307, PF-672, PF-C021, PF-C157, PF-C220, PF-C252, PF-C287, PF-5028,PF-168, PF-278, PF-283, PF-307, PF-527, PF-531, PF-547, PF-672, PF-0019,PF-C021, PF-C252, and PF-5028. In certain embodiments, the peptide iseffective to kill or inhibit the growth and/or proliferation of abacterium, where the amino acid sequence of the peptide comprises one ormore sequences selected from the group consisting of PF-006, PF-530,PF-531, PF-538, PF-C163, PF-C239, PF-C252, PF-C287, PF-006, PF-148,PF-283, PF-307, PF-322, PF-497, PF-499, PF-527, PF-531, PF-545, PF-547,PF-672, PF-5028, PF-522, PF-531, PF-538, PF-600, PF-606, PF-672,PF-C239, PF-C252, PF-006, PF-168, PF-209, PF-527, PF-545, PF-583,PF-606, PF-672, PF-C252, PF-5028, PF-5028, PF-C252, PF-531, PF-527,PF-5028, PF-C163, PF-C239, PF-C252, PF-C287, PF-278, PF-283, PF-527,PF-531, PF-583, PF-606, PF-672, PF-C163, PF-C252, PF-5028, PF-531,PF-547, PF-601, PF-0019, PF-C239, PF-C252, PF-5028, PF-437, PF-448,PF-511, PF-512, PF-520, PF-521, PF-523, PF-524, PF-525, PF-529, PF-600,and PF-606. In various embodiments the bacterium is selected from thegroup consisting of A. naeslundii, S. mutans, B. subtilis, MRSA, C.difficile, S. epidermidis, S. pneumoniae, E. faecalis, P. gingivalis, E.coli, P. aeruginosa, A. baumannii, and F. nucleatum.

In certain embodiments the peptide is effective to kill or inhibit thegrowth and/or proliferation of a gram positive bacterium, where theamino acid sequence of the peptide comprises one or more sequencesselected from the group consisting of PF-006, PF-148, PF-168, PF-209,PF-278, PF-283, PF-307, PF-322, PF-437, PF-448, PF-497, PF-499, PF-511,PF-512, PF-520, PF-521, PF-522, PF-523, PF-524, PF-525, PF-527, PF-529,PF-531, PF-538, PF-545, PF-547, PF-583, PF-600, PF-601, PF-606, PF-672,PF-0019, PF-C163, PF-C239, PF-C252, PF-C287, PF-S028. In certainembodiments the gram positive bacterium is selected from the groupconsisting of A. naeslundii, S. mutans, B. subtilis, MRSA, C. difficile,S. epidermidis, S. pneumoniae, and E. faecalis.

In certain embodiments the peptide is effective to kill or inhibit thegrowth and/or proliferation of gram negative bacterium where the aminoacid sequence of the peptide comprises one or more sequences selectedfrom the group consisting of PF-006, PF-527, PF-530, PF-531, PF-538,PF-600, PF-606, PF-C163, PF-C239, PF-C252, PF-C287, and PF-S028. Incertain embodiments the of gram negative bacterium is selected from thegroup consisting of P. gingivalis, E. coli, P. aeruginosa, A. baumannii,and F. nucleatum.

In various embodiments peptide(s) comprise all “L” amino acids, all “D”amino acids, or a mixture of “L” and “D” amino acids. In variousembodiments the peptide(s) are β peptides. The peptide(s) can optionallycomprise one or more protecting groups (e.g., an amide on the carboxylterminus and/or an acetyl on the amino terminus). In certain embodimentsthe peptide(s) are in a pharmaceutically acceptable carrier (e.g., acarrier suitable for administration via a route selected from the groupconsisting of topical administration, aerosol administration,administration via inhalation, oral administration, systemic IVapplication, ocular administration, rectal administration, etc.).

In various embodiments antimicrobial compositions are provided that areeffective to kill and/or to inhibit the growth and/or proliferation of amicroorganism and/or to inhibit the formation and/or growth and/ormaintenance of a biofilm comprising said microorganism. The compositionstypically comprise one or more peptides, the amino acid sequences of thepeptides comprising or consisting of one or more sequences selected fromthe group consisting of GSVIKKRRKRMSKKKHRKMLRRTRVQRRKLGK (PF-S028, SEQID NO:1), NYRLVNAIFSKIFKKKFIKF (PF-C252, SEQ ID NO:2),YIQFHLNQQPRPKVKKIKIFL (PF-531, SEQ ID NO:3),GSVIKKRRKRMAKKKHRKLLKKTRIQRRRAGK (PF-527, SEQ ID NO:4),MRFGSLALVAYDSAIKHSWPRPSSVRRLRM (PF-672, SEQ ID NO:5),FESKILNASKELDKEKKVNTALSFNSHQDFAKAYQNGKI (PF-606, SEQ ID NO:6),KGKSLMPLLKQINQWGKLYL (PF-C239, SEQ ID NO:7), WSRVPGHSDTGWKVWHRW (PF-547,SEQ ID NO:8), MGIIAGIIKFIKGLIEKFTGK (PF-006, SEQ ID NO:9),ILNKKPKLPLWKLGKNYFRRFYVLPTFLA (PF-C287, SEQ ID NO:10), RESKLIAMADMIRRRI(PF-545, SEQ ID NO:11), LDPLEPRIAPPGDRSHQGAPACHRDPLRGRSARDAER (PF-0019,SEQ ID NO:12), MPVSKKRYMLSSAYATALGICYGQVATDEKESEITAIPDLLDYLSVEEYLL(PF-C163, SEQ ID NO:13), LSLATFAKIFMTRSNWSLKRFNRL (PF-278, SEQ IDNO:14), MIRIRSPTKKKLNRNSISDWKSNTSGRFFY (PF-283, SEQ ID NO:15),MKRRRCNWCGKLFYLEEKSKEAYCCKECRKKAKKVKK (PF-307, SEQ ID NO:16),VLPFPAIPLSRRRACVAAPRPRSRQRAS (PF-168, SEQ ID NO:17),KNKKQTDILEKVKEILDKKKKTKSVGQKLY (PF-538, SEQ ID NO:18), SLQSQLGPCLHDQRH(PF-448, SEQ ID NO:19), WKRLWPARILAGHSRRRMRWMVVWRYFAAT (PF-C021, SEQ IDNO:20), KFQGEFTNIGQSYIVSASHMSTSLNTGK (PF-583, SEQ ID NO:21),TKKIELKRFVDAFVKKSYENYILERELKKLIKAINEELPTK (PF-600, SEQ ID NO:22),KFSDQIDKGQDALKDKLGDL (PF-525, SEQ ID NO:23), LSEMERRRLRKRA (PF-529, SEQID NO:24), RRGCTERLRRMARRNAWDLYAEHFY (PF-148, SEQ ID NO:25),SKFKVLRKIIIKEYKGELMLSIQKQR (PF-530, SEQ ID NO:26), FELVDWLETNLGKILKSKSA(PF-522, SEQ ID NO:27), LVLRICTDLFTFIKWTIKQRKS (PF-497, SEQ ID NO:28),VYSFLYVLVIVRKLLSMKKRIERL (PF-499, SEQ ID NO:29), GIVLIGLKLIPLLANVLR(PF-322, SEQ ID NO:30), VMQSLYVKPPLILVTKLAQQN (PF-511, SEQ ID NO:31),SFMPEIQKNTIPTQMK (PF-512, SEQ ID NO:32),LGLTAGVAYAAQPTNQPTNQPTNQPTNQPTNQPTNQPRW (PF-520, SEQ ID NO:33),CGKLLEQKNFFLKTR (PF-521, SEQ ID NO:34), ASKQASKQASKQASKQASKQASRSLKNHLL(PF-523, SEQ ID NO:35), PDAPRTCYHKPILAALSRIVVTDR (PF-524, SEQ ID NO:36),NYAVVSHT (PF-209, SEQ ID NO:37), ILVLLALQVELDSKFQY (PF-C157, SEQ IDNO:38), YVNYNQSFNSGW (PF-C220, SEQ ID NO:39), andFQKPFTGEEVEDFQDDDEIPTII (PF-437, SEQ ID NO:40).

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of a yeast or fungus, and thecomposition comprises one or more peptides, the amino acid sequences ofthe peptides comprising one or more sequences or inverse of thesequences independently selected from the group consisting of PF-148,PF-168, PF-448, PF-525, PF-527, PF-529, PF-531, PF-545, PF-672, PF-0019,PF-278, PF-307, PF-672, PF-C021, PF-C157, PF-C220, PF-C252, PF-C287,PF-5028, PF-168, PF-278, PF-283, PF-307, PF-527, PF-531, PF-547, PF-672,PF-0019, PF-C021, PF-C252, and PF-5028.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Aspergillus niger and the compositioncomprises one or more peptides, the amino acid sequences of the peptidescomprising one or more sequences or inverse of the sequencesindependently selected from the group consisting of PF-148, PF-168,PF-448, PF-525, PF-527, PF-529, PF-531, PF-545, PF-672, and PF-0019.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Candida albicans and the compositioncomprises one or more peptides, the amino acid sequences of the peptidescomprising one or more sequences or inverse of the sequencesindependently selected from the group consisting of PF-278, PF-307,PF-672, PF-C021, PF-C157, PF-C220, PF-C252, and PF-C287.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Malassezia furfur and the compositioncomprises a peptide comprising the amino acid sequence of PF-5028.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Trichophyton rubrum and thecomposition comprises one or more peptides, the amino acid sequences ofthe peptides comprising one or more sequences or inverse of thesequences independently selected from the group consisting of PF-168,PF-278, PF-283, PF-307, PF-527, PF-531, PF-547, PF-672, PF-0019,PF-C021, PF-C252, PF-S028.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of a bacterium and the compositioncomprises one or more peptides, the amino acid sequences of the peptidescomprising one or more sequences or inverse of the sequencesindependently selected from the group consisting of PF-006, PF-530,PF-531, PF-538, PF-C163, PF-C239, PF-C252, PF-C287, PF-006, PF-148,PF-283, PF-307, PF-322, PF-497, PF-499, PF-527, PF-531, PF-545, PF-547,PF-672, PF-S028, PF-522, PF-531, PF-538, PF-600, PF-606, PF-672,PF-C239, PF-C252, PF-006, PF-168, PF-209, PF-527, PF-545, PF-583,PF-606, PF-672, PF-C252, PF-S028, PF-S028, PF-C252, PF-531, PF-527,PF-S028, PF-C163, PF-C239, PF-C252, PF-C287, PF-278, PF-283, PF-527,PF-531, PF-583, PF-606, PF-672, PF-C163, PF-C252, PF-S028, PF-531,PF-547, PF-601, PF-0019, PF-C239, PF-C252, PF-S028, PF-437, PF-448,PF-511, PF-512, PF-520, PF-521, PF-523, PF-524, PF-525, PF-529, PF-600,and PF-606.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of a gram positive bacterium and thecomposition comprises one or more peptides, the amino acid sequences ofthe peptides comprising one or more sequences or inverse of thesequences independently selected from the group consisting of PF-006,PF-148, PF-168, PF-209, PF-278, PF-283, PF-307, PF-322, PF-437, PF-448,PF-497, PF-499, PF-511, PF-512, PF-520, PF-521, PF-522, PF-523, PF-524,PF-525, PF-527, PF-529, PF-531, PF-538, PF-545, PF-547, PF-583, PF-600,PF-601, PF-606, PF-672, PF-C019, PF-C163, PF-C239, PF-C252, PF-C287,PF-S028.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Actinomyces naeslundii and thecomposition comprises one or more peptides, the amino acid sequences ofthe peptides comprising one or more sequences or inverse of thesequences independently selected from the group consisting of PF-C163,PF-C239, PF-C252, and PF-C287.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Bacillus subtilis and the compositioncomprises one or more peptides, the amino acid sequences of the peptidescomprising one or more sequences or inverse of the sequencesindependently selected from the group consisting of PF-006, PF-148,PF-283, PF-307, PF-322, PF-497, PF-499, PF-527, PF-531, PF-545, PF-547,PF-672, and PF-S028.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Clostridium difficile and thecomposition comprises one or more peptides, the amino acid sequences ofthe peptides comprising one or more sequences or inverse of thesequences independently selected from the group consisting of PF-522,PF-531, and PF-538.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Enterococcus faecalis and thecomposition comprises one or more peptides comprising the amino acidsequence of PF-672.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Methicillin-resistant Staphylococcusaureus (MRSA) and the composition comprises one or more peptides, theamino acid sequences of the peptides comprising one or more sequences orinverse of the sequences independently selected from the groupconsisting of PF-006, PF-168, PF-209, PF-527, PF-545, PF-583, PF-606,PF-672, PF-C252, and PF-S028.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of S. epidermidis and the compositioncomprises one or more peptides, the amino acid sequences of the peptidescomprising one or more sequences or inverse of the sequencesindependently selected from the group consisting of PF-278, PF-283,PF-527, PF-531, PF-583, PF-606, PF-672, PF-C163, PF-C252, and PF-S028.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Streptococcus mutans and thecomposition comprises one or more peptides, the amino acid sequences ofthe peptides comprising one or more sequences or inverse of thesequences independently selected from the group consisting of PF-531,PF-547, PF-601, PF-0019, PF-C239, PF-C252, and PF-S028.

In certain embodiments the peptide composition is effective to kill orinhibit the growth and/or proliferation of Streptococcus pneumoniae andthe composition comprises one or more peptides, the amino acid sequencesof the peptides comprising one or more sequences or inverse of thesequences independently selected from the group consisting of PF-437,PF-448, PF-511, PF-512, PF-520, PF-521, PF-523, PF-524, PF-525, PF-529,PF-600, and PF-606.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of a gram negative bacterium and thecomposition comprises one or more peptides, the amino acid sequences ofthe peptides comprising one or more sequences or inverse of thesequences independently selected from the group consisting of PF-006,PF-527, PF-530, PF-531, PF-538, PF-600, PF-606, PF-C163, PF-C239,PF-C252, PF-C287, and PF-S028.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Acinetobacter baumannii and thecomposition comprises one or more peptides, the amino acid sequences ofthe peptides comprising one or more sequences or inverse of thesequences independently selected from the group consisting of PF-006,PF-530, PF-531, and PF-538.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Escherichia coli and the compositioncomprises one or more peptides, the amino acid sequences of the peptidescomprising one or more sequences or inverse of the sequencesindependently selected from the group consisting of PF-600, and PF-606.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Fusobacterium nucleatum and thecomposition comprises one or more peptides, the amino acid sequences ofthe peptides comprising one or more sequences s or inverse of thesequences elected from the group consisting of PF-C239, and PF-C252.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Pseudomonas aeruginosa and thecomposition comprises one or more peptides, the amino acid sequences ofthe peptides comprising one or more sequences or inverse of thesequences independently selected from the group consisting of PF-S028,PF-C252, PF-531, and PF-527.

In certain embodiments the composition is effective to kill or inhibitthe growth and/or proliferation of Porphyromonas gingivalis and thecomposition comprises one or more peptides, the amino acid sequences ofthe peptides comprising one or more sequences or inverse of thesequences independently selected from the group consisting of PF-S028,PF-C163, PF-C239, PF-C252, and PF-C287.

In various embodiments the one or more peptides comprising thecomposition comprises all “D” amino acids”, all “L” amino acids, or acombination of “D” and “L” amino acids. In various embodiments the oneor more peptides comprising the composition is a 0 peptide. In variousembodiments the one or more peptides comprising the composition compriseone or more protecting groups (e.g., an amide on the carboxyl terminusand/or an acetyl on the amino terminus). In certain embodiments thecomposition comprises a pharmaceutically acceptable carrier (e.g. acarrier suitable for administration via a route selected from the groupconsisting of topical administration, aerosol administration,administration via inhalation, oral administration, rectaladministration, etc.).

Methods are also provided for killing and/or inhibiting the growthand/or proliferation of a microorganism and/or inhibiting the formation,growth or maintenance of a biofilm comprising the microorganism. Themethods typically involve contacting the microorganism with one or moreantimicrobial peptides as described herein and/or a compositioncomprising one or more peptides, the amino acid sequences of thepeptides comprising one or more sequences or inverse of the sequencesindependently selected from the group consisting of PF-006, PF-530,PF-531, PF-538, PF-C163, PF-C239, PF-C252, PF-C287, PF-006, PF-148,PF-283, PF-307, PF-322, PF-497, PF-499, PF-527, PF-531, PF-545, PF-547,PF-672, PF-S028, PF-522, PF-531, PF-538, PF-600, PF-606, PF-672,PF-C239, PF-C252, PF-006, PF-168, PF-209, PF-527, PF-545, PF-583,PF-606, PF-672, PF-C252, PF-S028, PF-S028, PF-C252, PF-531, PF-527,PF-S028, PF-C163, PF-C239, PF-C252, PF-C287, PF-278, PF-283, PF-527,PF-531, PF-583, PF-606, PF-672, PF-C163, PF-C252, PF-5028, PF-531,PF-547, PF-601, PF-0019, PF-C239, PF-C252, PF-S028, PF-437, PF-448,PF-511, PF-512, PF-520, PF-521, PF-523, PF-524, PF-525, PF-529, PF-600,and PF-606, e.g., as described herein (see, for example, descriptionsupra and AMPs and compositions recited in the claims). In certainembodiments the contacting comprises contacting the microorganism withan amount sufficient to kill or to inhibit the growth or proliferationof said microorganism.

In various embodiments methods of disinfecting a surface are alsoprovided. The methods typically involve contacting the surface with oneor more antimicrobial peptides as described herein and/or a compositioncomprising one or more peptides, the amino acid sequences of thepeptides comprising one or more sequences or inverse of the sequencesindependently selected from the group consisting of PF-006, PF-530,PF-531, PF-538, PF-C163, PF-C239, PF-C252, PF-C287, PF-006, PF-148,PF-283, PF-307, PF-322, PF-497, PF-499, PF-527, PF-531, PF-545, PF-547,PF-672, PF-S028, PF-522, PF-531, PF-538, PF-600, PF-606, PF-672,PF-C239, PF-C252, PF-006, PF-168, PF-209, PF-527, PF-545, PF-583,PF-606, PF-672, PF-C252, PF-S028, PF-S028, PF-C252, PF-531, PF-527,PF-5028, PF-C163, PF-C239, PF-C252, PF-C287, PF-278, PF-283, PF-527,PF-531, PF-583, PF-606, PF-672, PF-C163, PF-C252, PF-S028, PF-531,PF-547, PF-601, PF-0019, PF-C239, PF-C252, PF-S028, PF-437, PF-448,PF-511, PF-512, PF-520, PF-521, PF-523, PF-524, PF-525, PF-529, PF-600,and PF-606, e.g., as described herein (see, for example, descriptionsupra and AMPs and compositions recited in the claims). In certainembodiments the surface comprises a surface of a prosthesis and/ormedical implant, and/or the surface comprises a surface of a medicaldevice, or a surface of a plant or foodstuff. In certain embodiments thepeptide(s) are combined with, or used in conjunction with, a seconddisinfectant selected from the group consisting of other antimicrobialagent is a disinfectant selected from the group consisting of aceticacid, phosphoric acid, citric acid, lactic, formic, propionic acid,hydrochloric acid, sulfuric acid, nitric acid, sodium hydroxide,potassium hydroxide, sodium carbonate, ammonium hydroxide, ethylalcohol, isopropyl alcohol, phenol, formaldehyde, glutaraldehyde,hypochlorites, chlorine dioxide, sodium dichloroisocyanurate,chloramine-T, iodine, povidone-iodine, chlorhexidine, hydrogen peroxide,peracetic acid, and benzalkonium chloride.

Also provided is the use one or more antimicrobial peptides as describedherein and/or a composition comprising one or more peptides, the aminoacid sequences of the peptides comprising one or more sequences orinverse of the sequences independently selected from the groupconsisting of PF-006, PF-530, PF-531, PF-538, PF-C163, PF-C239, PF-C252,PF-C287, PF-006, PF-148, PF-283, PF-307, PF-322, PF-497, PF-499, PF-527,PF-531, PF-545, PF-547, PF-672, PF-S028, PF-522, PF-531, PF-538, PF-600,PF-606, PF-672, PF-C239, PF-C252, PF-006, PF-168, PF-209, PF-527,PF-545, PF-583, PF-606, PF-672, PF-C252, PF-S028, PF-S028, PF-C252,PF-531, PF-527, PF-S028, PF-C163, PF-C239, PF-C252, PF-C287, PF-278,PF-283, PF-527, PF-531, PF-583, PF-606, PF-672, PF-C163, PF-C252,PF-S028, PF-531, PF-547, PF-601, PF-0019, PF-C239, PF-C252, PF-S028,PF-437, PF-448, PF-511, PF-512, PF-520, PF-521, PF-523, PF-524, PF-525,PF-529, PF-600, and PF-606, e.g., as described herein (see, for example,description supra and AMPs and compositions recited in the claims) inthe manufacture of a medicament for killing and/or inhibiting the growthand/or proliferation of a microorganism. In certain embodiments themicroorganism is a yeast or fungus and the peptide or composition is apeptide or composition comprising one or more AMPs identified herein askilling a yeast or fungus. In certain embodiments the microorganism is abacterium and the peptide or composition is a peptide or compositioncomprising one or more AMPs identified herein as killing a bacterium. Incertain embodiments the microorganism is a gram positive bacterium andthe peptide or composition is a peptide or composition comprising one ormore AMPs identified herein as killing a gram positive bacterium. Incertain embodiments the microorganism is a gram negative bacterium andthe peptide or composition is a peptide or composition comprising one ormore AMPs identified herein as killing a gram negative bacterium.

DEFINITIONS

The term “peptide” as used herein refers to a polymer of amino acidresidues typically ranging in length from 2 to about 50 or about 60residues (in certain instances up to about 100 residues). In certainembodiments the peptide ranges in length from about 2, 3, 4, 5, 7, 9,10, or 11 residues to about 51, 50, 45, 40, 45, 30, 25, 20, or 15residues. In certain embodiments the peptide ranges in length from about8, 9, 10, 11, or 12 residues to about 15, 20, 25, 35, 50, or 51residues. In certain embodiments the amino acid residues comprising thepeptide are “L-form” amino acid residues, however, it is recognized thatin various embodiments, “D” amino acids can be incorporated into thepeptide. Peptides also include amino acid polymers in which one or moreamino acid residues is an artificial chemical analogue of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers. In addition, the term applies to aminoacids joined by a peptide linkage or by other, “modified linkages”(e.g., where the peptide bond is replaced by an α-ester, a β-ester, athioamide, phosphonamide, carbomate, hydroxylate, and the like (see,e.g., Spatola (1983) Chem. Biochem. Amino Acids and Proteins 7:267-357), where the amide is replaced with a saturated amine (see, e.g.,Skiles et al., U.S. Pat. No. 4,496,542; and Kaltenbronn et al., (1990)Pp. 969-970 in Proc. 11th American Peptide Symposium, ESCOM SciencePublishers, The Netherlands, and the like)).

The term “residue”” as used herein refers to natural, synthetic, ormodified amino acids. Various amino acid analogues include, but are notlimited to 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine(beta-aminopropionic acid), 2-aminobutyric acid, 4-aminobutyric acid,piperidinic acid, 6-aminocaproic acid, 2-aminoheptanoic acid,2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4diaminobutyric acid, desmosine, 2,2′-diaminopimelic acid,2,3-diaminopropionic acid, n-ethylglycine, n-ethylasparagine,hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline,isodesmosine, allo-isoleucine, n-methylglycine, sarcosine,n-methylisoleucine, 6-n-methyllysine, n-methylvaline, norvaline,norleucine, ornithine, and the like. These modified amino acids areillustrative and not intended to be limiting.

“β-peptides” comprise of “β amino acids”, which have their amino groupbonded to the β carbon rather than the α-carbon as in the 20 standardbiological amino acids. The only commonly naturally occurring β aminoacid is β-alanine.

Peptoids, or N-substituted glycines, are a specific subclass ofpeptidomimetics. They are closely related to their natural peptidecounterparts, but differ chemically in that their side chains areappended to nitrogen atoms along the molecule's backbone, rather than tothe α-carbons (as they are in natural amino acids).

The terms “conventional” and “natural” as applied to peptides hereinrefer to peptides, constructed only from the naturally-occurring aminoacids: Ala, Cys, Asp, Glu, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,Pro, Gln, Arg, Ser, Thr, Val, Tip, and Tyr. A compound of the invention“corresponds” to a natural peptide if it elicits a biological activity(e.g., antimicrobial activity) related to the biological activity and/orspecificity of the naturally occurring peptide. The elicited activitymay be the same as, greater than or less than that of the naturalpeptide. In general, such a peptoid will have an essentiallycorresponding monomer sequence, where a natural amino acid is replacedby an N-substituted glycine derivative, if the N-substituted glycinederivative resembles the original amino acid in hydrophilicity,hydrophobicity, polarity, etc. The following are illustrative, butnon-limiting N-substituted glycine replacements:N-(1-methylprop-1-yl)glycine substituted for isoleucine (Ile),N-(prop-2-yl)glycine for valine (Val), N-benzylglycine for phenylanlaine(Phe), N-(2-hydroxyethyl)glycine for serine (Ser), and the like. Incertain embodiments substitutions need not be “exact”. Thus for example,in certain embodiments N-(2-hydroxyethyl)glycine may substitute for Ser,Thr, Cys, and/or Met; N-(2-methylprop-1-yl)glycine may substitute forVal, Leu, and/or Ile. In certain embodiments N-(2-hydroxyethyl)glycinecan be used to substitute for Thr and Ser, despite the structuraldifferences: the side chain in N-(2-hydroxyethyl)glycine is onemethylene group longer than that of Ser, and differs from Thr in thesite of hydroxy-substitution. In general, one may use anN-hydroxyalkyl-substituted glycine to substitute for any polar aminoacid, an N-benzyl- or N-aralkyl-substituted glycine to replace anyaromatic amino acid (e.g., Phe, Trp, etc.), an N-alkyl-substitutedglycine such as N-butylglycine to replace any nonpolar amino acid (e.g.,Leu, Val, Ile, etc.), and an N-(aminoalkyl)glycine derivative to replaceany basic polar amino acid (e.g., Lys and Arg).

Where an amino acid sequence is provided herein, L-, D-, or beta aminoacid versions of the sequence are also contemplated as well as retro,inversion, and retro-inversion isoforms. In addition, conservativesubstitutions (e.g., in the binding peptide, and/or antimicrobialpeptide, and/or linker peptide) are contemplated. Non-protein backbones,such as PEG, alkane, ethylene bridged, ester backbones, and otherbackbones are also contemplated. Also fragments ranging in length fromabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 amino acids up to the full length minus one amino acid ofthe peptide are contemplated where the fragment retains at least 50%,preferably at least 60% 70% or 80%, more preferably at least 90%, 95%,98%, 99%, or at least 100% of the activity (e.g., binding specificityand/or avidity, antimicrobial activity, etc.) of the full length peptideare contemplated.

In certain embodiments, conservative substitutions of the amino acidscomprising any of the sequences described herein are contemplated. Invarious embodiments one, two, three, four, or five different residuesare substituted. The term “conservative substitution” is used to reflectamino acid substitutions that do not substantially alter the activity(e.g., antimicrobial activity and/or specificity) of the molecule.Typically conservative amino acid substitutions involve substitution oneamino acid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). Certain conservative substitutions include“analog substitutions” where a standard amino acid is replaced by anon-standard (e.g., rare, synthetic, etc) amino acid differing minimallyfrom the parental residue. Amino acid analogs are considered to bederived synthetically from the standard amino acids without sufficientchange to the structure of the parent, are isomers, or are metaboliteprecursors. Examples of such “analog substitutions include but are notlimited to 1) Lys-Orn, 2) Leu-Norleucine, 3) Lys-Lys[TFA], 4)Phe-Phe[Gly], and 5) 6-amino butylglycine-4-amino hexylglycine, wherePhe[gly] refers to phenylglycine (a Phe derivative with a H rather thanCH₃ component in the R group), and Lys[TFA] refers to a Lys where anegatively charged ion (e.g., TFA) is attached to the amine R group.Other conservative substitutions include “functional substitutions”where the general chemistries of the two residues are similar, and canbe sufficient to mimic or partially recover the function of the nativepeptide. Strong functional substitutions include, but are not limitedto 1) Gly/Ala, 2) Arg/Lys, 3) Ser/Tyr/Thr, 4) Leu/Ile/Val, 5) Asp/Glu,6) Gln/Asn, and 7) Phe/Trp/Tyr, while other functional substitutionsinclude, but are not limited to 8) Gly/Ala/Pro, 9) Tyr/His, 10)Arg/Lys/His, 11) Ser/Thr/Cys, 12) Leu/Ile/Val/Met, and 13) Met/Lys(special case under hydrophobic conditions). Various “broad conservativesubstations” include substitutions where amino acids replace other aminoacids from the same biochemical or biophysical grouping. This issimilarity at a basic level and stems from efforts to classify theoriginal 20 natural amino acids. Such substitutions include 1) nonpolarside chains: Gly/Ala/Val/Leu/Ile/Met/Pro/Phe/Trp, and/or 2) unchargedpolar side chains Ser/Thr/Asn/Gln/Tyr/Cys. In certain embodimentsbroad-level substitutions can also occur as paired substitutions. Forexample, Any hydrophilic neutral pair [Ser, Thr, Gln, Asn, Tyr,Cys]+[Ser, Thr, Gln, Asn, Tyr, Cys] can may be replaced by acharge-neutral charged pair [Arg, Lys, His]+[Asp, Glu]. The followingsix groups each contain amino acids that, in certain embodiments, aretypical conservative substitutions for one another: 1) Alanine (A),Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3)Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K), Histidine(H); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Where amino acidsequences are disclosed herein, amino acid sequences comprising, one ormore of the above-identified conservative substitutions are alsocontemplated.

Antimicrobial peptides described herein also include “compoundantimicrobial peptides” or “compound AMP(s)” that are constructscomprising two or more AMPs joined together thus forming a molecule withmultiple AMP domains (that are the same or different). The AMPs can bejoined directly or through a linker. They can be chemically conjugatedor, where joined directly together or through a peptide linker cancomprise a fusion protein.

In certain embodiments antimicrobial peptides compromising at least 80%,preferably at least 85% or 90%, and more preferably at least 95% or 98%or 99% sequence identity with any of the sequences described herein arealso contemplated. The terms “identical” or percent “identity,” refer totwo or more sequences that are the same or have a specified percentageof amino acid residues that are the same, when compared and aligned formaximum correspondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. With respect to thepeptides of this invention sequence identity is determined over the fulllength of the peptide. For sequence comparison, typically one sequenceacts as a reference sequence, to which test sequences are compared. Whenusing a sequence comparison algorithm, test and reference sequences areinput into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated. Thesequence comparison algorithm then calculates the percent sequenceidentity for the test sequence(s) relative to the reference sequence,based on the designated program parameters. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by thehomology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48: 443, by the search for similarity method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci., USA, 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection.

The term “specificity” when used with respect to the antimicrobialactivity of a peptide indicates that the peptide preferentially inhibitsgrowth and/or proliferation and/or kills a particular microbial speciesas compared to other related and/or unrelated microbes. In certainembodiments the preferential inhibition or killing is at least 10%greater (e.g., LD₅₀ is 10% lower), preferably at least 20%, 30%, 40%, or50%, more preferably at least 2-fold, at least 5-fold, or at least10-fold greater for the target species.

“Treating” or “treatment” of a condition as used herein may refer topreventing the condition, slowing the onset or rate of development ofthe condition, reducing the risk of developing the condition, preventingor delaying the development of symptoms associated with the condition,reducing or ending symptoms associated with the condition, generating acomplete or partial regression of the condition, or some combinationthereof.

The term “consisting essentially of” when used with respect to anantimicrobial peptide (AMP) or AMP motif as described herein, indicatesthat the peptide or peptides encompassed by the library or variants,analogues, or derivatives thereof possess substantially the same orgreater antimicrobial activity and/or specificity as the referencedpeptide. In certain embodiments substantially the same or greaterantimicrobial activity indicates at least 80%, preferably at least 90%,and more preferably at least 95% of the anti microbial activity of thereferenced peptide(s) against a particular bacterial species (e.g., S.mutans).

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies, including, but are not limited to, Fab′₂,IgG, IgM, IgA, scFv, dAb, nanobodies, unibodies, and diabodies.

In certain embodiments antibodies and fragments of the present inventioncan be bispecific. Bispecific antibodies or fragments can be of severalconfigurations. For example, bispecific antibodies may resemble singleantibodies (or antibody fragments) but have two different antigenbinding sites (variable regions). In various embodiments bispecificantibodies can be produced by chemical techniques (Kranz et al. (1981)Proc. Natl. Acad. Sci., USA, 78: 5807), by “polydoma” techniques (see,e.g., U.S. Pat. No. 4,474,893), or by recombinant DNA techniques. Incertain embodiments bispecific antibodies of the present invention canhave binding specificities for at least two different epitopes, at leastone of which is an epitope of a microbial organism. The microbialbinding antibodies and fragments can also be heteroantibodies.Heteroantibodies are two or more antibodies, or antibody bindingfragments (e.g., Fab) linked together, each antibody or fragment havinga different specificity.

The term “STAMP” refers to Specifically Targeted Anti-MicrobialPeptides. In various embodiments, a STAMP comprises one or more peptidetargeting moieties attached to one or more antimicrobial moieties (e.g.,antimicrobial peptides (AMPs)). An MH-STAMP is a STAMP bearing two ormore targeting domains (i.e., a multi-headed STAMP).

The terms “isolated” “purified” or “biologically pure” refer to materialwhich is substantially or essentially free from components that normallyaccompany it as found in its native state. In the case of a peptide, anisolated (naturally occurring) peptide is typically substantially freeof components with which it is associated in the cell, tissue, ororganism. The term isolated also indicates that the peptide is notpresent in a phage display, yeast display, or other peptide library.

In various embodiments the amino acid abbreviations shown in Table 1 areused herein.

TABLE 1 Various amino acid abbreviations. Abbreviation Name 3 Letter 1Letter Alanine Ala A βAlanine (NH₂—CH₂—CH₂—COOH) βAla Arginine Arg RAsparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu EGlutamine Gln Q Glycine Gly G Histidine His H Homoserine Hse —Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Methioninesulfoxide Met (O) — Methionine methylsulfonium Met (S—Me) — NorleucineNle — Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V episilon-aminocaproic acidAhx J (NH²—(CH₂)₅—COOH) 4-aminobutanoic acid gAbu (NH₂—(CH₂)₃—COOH)tetrahydroisoquinoline-3- O carboxylic acidLys(N(epsilon)-trifluoroacetyl) K[TFA] α-aminoisobutyric acid Aib B

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Killing kinetics of PF-S028 L and D versions against M. furfurwere determined using standard time-kill methods previously detailed(Eckert et al. (2006) Antimicrob Agents Chemother., 50: 1480-1488).Briefly, a log phase culture of M. furfur ATCC 14521 was diluted to 10⁶cells/ml in ATCC medium 1072. To the reaction tubes 5 μM of eitherPF-S028 L or PF-S028 D were added. A reaction tube to which 4 μA of 50%methanol served as the negative control. Reaction tubes were stored at30° C. and at indicated intervals an aliquot was removed from thereaction tube and placed in a recovery tube where peptide was removed bydilution. An aliquot from the recovery tube was plated on ATCC medium1072 agar plates and incubated at 30° C. until visible colonies formed.Colony forming units were calculated for the negative control (nopeptide), PF-S028 L, and PF-S028 D treated cultures. After 15 min oftreatment, PF-S028 D killed over 99% of the M. furfur. PF-S028 L killed39%.

DETAILED DESCRIPTION

In various embodiments novel peptides having antimicrobial activityagainst certain bacteria, fungi, and/or yeasts are provided. Whenexploited for their antimicrobial activity, the novel antimicrobialpeptides described herein (see, e.g., Table 3) can be used to inhibitthe growth and/or proliferation of a microbial species and/or the growthand/formation and/or maintenance of a biofilm comprising the microbialspecies. In various embodiments the peptides can be formulatedindividually, in combination with each other, in combination with otherantimicrobial peptides, and/or in combination with various antibacterialagents to provide antimicrobial reagents and/or pharmaceuticals.

Accordingly, in certain embodiments this invention provides peptideshaving antimicrobial activity, compositions comprising the peptides,methods of using the peptides (or compositions thereof) to inhibit thegrowth of or kill a wide variety of microbial targets and methods ofusing the peptides (or compositions thereof) to treat or preventmicrobial infections and diseases related thereto in both plants andanimals.

The various peptides described herein exhibit antimicrobial activity,being biostatic or biocidal against a certain microbial targets,including but not limited to, Gram-negative bacteria such asAcinetobacter baumannii, Escherichia coli, Fusobacterium nucleatum,Pseudomonas aeruginosa, Porphyromonas gingivalis; Gram-positive bacteriasuch as Actinomyces naeslundii, Bacillus subtilis, Clostridiumdifficile, Enterococcus faecalis, Staphylococcus aureus (and MRSA), S.epidermidis, Streptococcus mutans, Streptococcus pneumoniae; and yeastor fungi such as Aspergillus niger, Candida albicans, Malassezia furfur,and Trichophyton rubrum (see, e.g., Table 2). Significantly, variouspeptides described herein are biostatic or biocidal against clinicallyrelevant pathogens exhibiting multi-drug resistance such as, forexample, methicillin-resistant Staphylococcus aureus (“MRSA”).

TABLE 2 Illustrative target microorganisms and associated pathology.Acinetobacter baumannii Pathogenic gram-negative bacillus that is (A.baumannii) naturally sensitive to relatively few antibiotics.Actinomyces naeslundii Gram positive rod shaped bacteria that (A.naeslundii) occupy the oral cavity and are implicated in periodontaldisease and root caries. Aspergillus niger A fungal infection that oftencauses a (A. niger) black mould to appear on some fruit and vegetablesbut may also infect humans through inhalation of fungal spores. Bacillussubtilis Gram-positive, catalase-positive (B. subtilis) bacterium.Candida albicans Causal agent of opportunistic oral and (C. albicans)genital fungal infections in humans. Clostridium difficile Agram-positive, anaerobic, spore-forming (C. difficile) bacillus that isresponsible for the development of antibiotic-associated diarrhea andcolitis. Corynebacterium jeikium Gram-positive, normal skin flora (C.jeikium) associated with malodor. Opportunistic pathogen. Escherichiacoli Gram negative rod-shaped bacterium (E. coli) commonly found in thelower intestine of warm-blooded organisms. Certain strains cause seriousfood poisoning in humans. Enterococcus faecalis Gram-positive commensalbacterium and (E. faecalis) opportunistic pathogen. Fusobacteriumnucleatum Gram negative schizomycetes bacterium (F. nucleatum) oftenseen in necrotic tissue and implicated, but not conclusively, with otherorganisms in the causation and perpetuation of periodontal disease.Malassezia furfur yeast—cutaneous pathogen (M. furfur)Methicillin-resistant Any strain of Staphylococcus aureus Staphylococcusaureus bacteria (gram positive) that is (MRSA) resistant to a one ormore members of a large group of antibiotics called the beta-lactams.Responsible for skin and systemic infections. Pseudomonas aeruginosaGram-negative rod, common opportunistic P. aeruginosa pathogen.Responsible for lung, ear, and skin infections. Second most prevalentsource of burn wound infections. Porphyromonas gingivalis Belongs to thegenus Bacteroides and is a (P. gingivalis) non-motile, gram-negative,rod-shaped, anaerobic pathogenic bacterium (periodontal disease) S.epidermidis Gram-positive, coagulase-negative cocci. (S. epidermidis)Nosocomial pathogen associated with infection (biofilm) of implantedmedical devices. Streptococcus mutans Gram-positive, facultativelyanaerobic (S. mutans) bacterium commonly found in the human oral cavityand is the primary contributor to tooth decay StreptococcusGram-positive, alpha-hemolytic, bile pneumoniae soluble aerotolerantanaerobe. Causal (S. pneumoniae) agent for streptococcal pneumonia.Trichophyton rubrum Most common cause of athlete's foot, jock (T.rubrum) itch and ringworm.

In certain embodiments, the antimicrobial peptide is attached to anopsinon or lysosome uptake or internalization signal to facilitatecellular uptake and killing of intracellular microorganisms.

I. Antimicrobial Peptides.

In certain embodiments the antimicrobial peptides include peptidescomprising or consisting of one or more of the amino acid sequencesshown in Table 3 (SEQ ID NOs:1-40). In various embodiments the peptidesinclude peptides comprising or consisting of the retro, inverso,retro-inverso, and/or beta form of one or more of the amino acidsequences shown in Table 3 (SEQ ID NOs:1-40). Also contemplated arecircular permutations of these sequences as well as peptides comprisingor consisting of the retro, inverso, retro-inverso, and/or beta form ofsuch circular permutations.

It will also be recognized, that in certain embodiments, any peptide orcompound AMP described herein can be circularized.

In various embodiments the peptides can optionally bear one or moreprotecting groups, e.g., and the amino and/or carboxyl termini, and/oron side chains.

Also contemplated are peptides comprising one, two, three four, or fiveconservative substitutions of these amino acid sequences.

TABLE 3  Illustrative antimicrobial peptide sequences, target organismsand the MIC (lowest concentration of an antibiotic which willinhibit the (in vitro) growth) of the listed target organism(s). MIC SEQName Amino Acid Sequence Organisms (μM) ID No. PF-S028GSVIKKRRKRMSKKKHRKMLRRTR P. aeruginosa 50 1 VQRRKLGK P. gingivalis 25T. rubrum 50 M. furfur 2.5 B. subtilis 12.5 C. jeikeium 6.25 MRSA 50S. epidermidis 25 S. mutans 50 PF-C252 NYRLVNAIFSKIFKKKFIKFP. aeruginosa 50 2 P. gingivalis 25 C. albicans 25 T. rubrum 50A. naeslundii 25 F. nucleatum 25 MRSA 50 S. epidermidis 25 S. mutans12.5 PF-531 YIQFHLNQQPRPKVKKIKIFL A. baumannii 25 3 P. aeruginosa 50T. rubrum 50 A. niger 25 B. subtilis 25 C. difficile 12.5 C. jeikeium6.25 S. epidermidis 50 S. mutans 12.5 PF-527 GSVIKKRRKRMAKKKHRKLLKKTRIP. aeruginosa 50 4 QRRRAGK T. rubrum 25 A. niger 50 B. subtilis 12.5C. jeikeium 6.25 MRSA 50 S. epidermidis 25 PF-672MRFGSLALVAYDSAIKHSWPRPSSV C. albicans 1.56 5 RRLRM T. rubrum 0.78A. niger 3 B. subtilis 0.78 E. faecalis 3.13 MRSA 1.56 S. epidermidis0.39 PF-606 FESKILNASKELDKEKKVNTALSFNS E. coli 50 6 HQDFAKAYQNGKI MRSA50 S. epidermidis 50 S. mutans 50 S. pneumoniae 50 PF-C239KGKSLMPLLKQINQWGKLYL P. gingivalis 50 7 A. naeslundii 25 F. nucleatum 50S. mutans 50 PF-547 WSRVPGHSDTGWKVWHRW T. rubrum 25 8 B. subtilis 25S. mutans 12.5 PF-006 MGIIAGIIKFIKGLIEKFTGK A. baumannii 50 9B. subtilis 25 MRSA 50 PF-C287 ILNKKPKLPLWKLGKNYFRRFYVLP P. gingivalis50 10 TFLA C. albicans 50 A. naeslundii 25 PF-545 RESKLIAMADMIRRRIA. niger 50 11 B. subtilis 25 MRSA 50 PF-C019 LDPLEPRIAPPGDRSHQGAPACHRDPT. rubrum 50 12 LRGRSARDAER A. niger 50 S. mutans 25 PF-C163MPVSKKRYMLSSAYATALGICYGQ P. gingivalis 50 13 VATDEKESEITAIPDLLDYLSVEEYLA. naeslundii 50 L S. epidermidis 50 PF-278 LSLATFAKIFMTRSNWSLKRFNRLC. albicans 50 14 T. rubrum 50 S. epidermidis 50 PF-283MIRIRSPTKKKLNRNSISDWKSNTSG T. rubrum 50 15 RFFY B. subtilis 50S. epidermidis 50 PF-307 MKRRRCNWCGKLFYLEEKSKEAYC C. albicans 50 16CKECRKKAKKVKK T. rubrum 50 B. subtilis 50 PF-168VLPFPAIPLSRRRACVAAPRPRSRQR T. rubrum 50 17 AS A. niger 50 MRSA 50 PF-538KNKKQTDILEKVKEILDKKKKTKSV A. baumannii 25 18 GQKLY C. difficile 25PF-448 SLQSQLGPCLHDQRH A. niger 25 19 S. pneumoniae 50 PF-C021WKRLWPARILAGHSRRRMRWMVV C. albicans 50 20 WRYFAAT T. rubrum 50 PF-583KFQGEFTNIGQSYIVSASHMSTSLNT MRSA 50 21 GK S. epidermidis 50 PF-600TKKIELKRFVDAFVKKSYENYILERE E. coli 50 22 LKKLIKAINEELPTK S. pneumoniae50 PF-525 KFSDQIDKGQDALKDKLGDL A. niger 50 23 S. pneumoniae 50 PF-529LSEMERRRLRKRA A. niger 50 24 S. pneumoniae 50 PF-148RRGCTERLRRMARRNAWDLYAEHF A. niger 50 25 Y B. subtilis 50 PF-530SKFKVLRKIIIKEYKGELMLSIQKQR A. baumannii 25 26 PF-522FELVDWLETNLGKILKSKSA C. difficile 25 27 PF-497 LVLRICTDLFTFIKWTIKQRKSB. subtilis 50 28 PF-499 VYSFLYVLVIVRKLLSMKKRIERL B. subtilis 50 29PF-322 GIVLIGLKLIPLLANVLR B. subtilis 50 30 PF-511 VMQSLYVKPPLILVTKLAQQNS. pneumoniae 50 31 PF-512 SFMPEIQKNTIPTQMK S. pneumoniae 50 32 PF-520LGLTAGVAYAAQPTNQPTNQPTNQP S. pneumoniae 50 33 TNQPTNQPTNQPRW PF-521CGKLLEQKNFFLKTR S. pneumoniae 50 34 PF-523 ASKQASKQASKQASKQASKQASRSS. pneumoniae 50 35 LKNHLL PF-524 PDAPRTCYHKPILAALSRIVVTDR S. pneumoniae50 36 PF-209 NYAVVSHT MRSA 50 37 PF-C157 ILVLLALQVELDSKFQY C. albicans50 38 PF-C220 YVNYNQSFNSGW C. albicans 50 39 PF-437FQKPFTGEEVEDFQDDDEIPTII S. pneumoniae 50 40

In certain embodiments, the amino acid sequence of the antimicrobialpeptides comprises or consists of a single amino acid sequence, e.g., aslisted above. In certain embodiments the amino acid sequence of theantimicrobial peptides comprises two copies, three copies, four copies,five copies six copies or more of one or more of the amino acidsequences listed above. Thus, compound antimicrobial constructs arecontemplated where the construct comprises multiple domains each havingantimicrobial activity. The AMP domains comprising such a construct canbe the same or different. In certain embodiments the construct comprisesat least 2, at least 3, at least 4, at least 5, at least 6, at least 7,or at least 8 different AMP domains each domain comprising a differentAMP sequence.

Various AMP domains comprising such a construct can be joined directlyto each other or two or more of such domains can be attached to eachother via a linker. An illustrative, but non-limiting, list of suitablelinkers is provided in Table 4.

TABLE 4 Illustrative peptide and non-peptide linkers for joining AMP domains and/or forjoining AMPs and/or compound AMPs to one or more targeting or other moieties.Linker SEQ ID NO: AAA 41 GGG 42 GGGG 43 GGGGG 44 SGG 45 GGSGGS 46 SAT 47PYP 48 PSPSP 49 ASA 50 ASASA 51 PSPSP 52 KKKK 53 RRRR 54 Gly₄Ser 55(Gly₄Ser)₂ 56 (Gly₄Ser)₃ 57 (Gly₄Ser)₄ 58 (Gly₄Ser)₅ 59 (Gly₄Ser)₆ 602-nitrobenzene or O-nitrobenzyl Nitropyridyl disulfideDioleoylphosphatidylethanolamine (DOPE) S-acetylmercaptosuccinic acid1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA)β-glucuronide and β-glucuronide variants Poly(alkylacrylic acid)Benzene-based linkers (for example: 2,5-Bis(hexyloxy)-1,4-bis[2,5-bis(hexyloxy)-4-formyl-phenylenevinylene]benzene) and like.Disulfide linkagesPoly(amidoamine) or like dendrimers linking multiple target andkilling peptides in one molecule Carbon nanotubesHydrazone and hydrazone variant linkers PEG of any chain lengthSuccinate, formate, acetate butyrate, other like organic acidsAldols, alcohols, or enols PeroxidesAlkane or alkene groups of any chain lengthOne or more porphyrin or dye molecules containing free amide andcarboxylic acid groupsOne or more DNA or RNA nucleotides, including polyamine andpolycarboxyl-containing variantsInulin, sucrose, glucose, or other single, di or polysaccharidesLinoleic acid or other polyunsaturated fatty acidsVariants of any of the above linkers containing halogen or thiol groups(allamino-acid-based linkers could be L, D, β, or other forms)Thus, in certain embodiments, two or more AMP domains comprising acompound AMP construct are chemically conjugated together.

In certain embodiments the two or more AMP domains comprising the AMPconstruct are joined by a peptide linker. Where all the AMP domains areattached directly to each other or are joined by peptide linkers, theentire construct can be provided as a single-chain peptide (fusionprotein).

In various embodiments, the antimicrobial peptides described hereincomprise one or more of the amino acid sequences shown in Table 3(and/or the retro, inverso, retroinverso, etc. forms of such sequences).In certain embodiments the peptides range in length up to about 100amino acids in length, preferably up to about 80, about 70, about 60, orabout 51 amino acids in length. In certain embodiments the peptidesrange in length from about 8 amino acids up to about 100 amino acids 80amino acids, 60 amino acids or about 51 amino acids in length. Incertain embodiments the peptides range in length from about 8 up toabout 50, 40, 30, 20, 15, 15, 13, or 12 amino acids in length.

As shown in Tables 3 and 5, the various amino acid sequences describedherein are effective against particular microorganisms. The range ofactivity of the peptides or compositions comprising such peptides can beincreased by including amino acid sequences effective against differentmicroorganisms either as separate components and/or as multiple domainswithin a single construct.

TABLE 5 Illustrative target microorganisms and peptides effectiveagainst that target microorganism. Organism Peptide Gram PositiveBacteria: A. naeslundii PF-C163 PF-C239 PF-C252 PF-C287 S. mutans PF-531PF-547 PF-601 PF-C019 PF-C239 PF-C252 PF-S028 B. subtilis PF-006 PF-148PF-283 PF-307 PF-322 PF-497 PF-499 PF-527 PF-531 PF-545 PF-547 PF-672PF-S028 MRSA PF-006 PF-168 PF-209 PF-527 PF-545 PF-583 PF-606 PF-672PF-C252 PF-S028 C. jeikium PF-531 PF-S028 PF-527 C. difficile PF-522PF-531 PF-538 S. epidermidis PF-278 PF-283 PF-527 PF-531 PF-583 PF-606PF-672 PF-C163 PF-C252 PF-S028 S. pneumoniae PF-437 PF-448 PF-511 PF-512PF-520 PF-521 PF-523 PF-524 PF-525 PF-529 PF-600 PF-606 E. faecalisPF-672 Gram Negative Bacteria: P. gingivalis PF-S028 PF-C163 PF-C239PF-C252 PF-C287 E. coli PF-600 PF-606 P. aeruginosa PF-S028 PF-C252PF-531 PF-527 A. baumannii PF-006 PF-530 PF-531 PF-538 F. nucleatumPF-C239 PF-C252 Yeast/Fungi: A. niger PF-148 PF-168 PF-448 PF-525 PF-527PF-529 PF-531 PF-545 PF-672 PF-C019 C. albicans PF-278 PF-307 PF-672PF-C021 PF-C157 PF-C220 PF-C252 PF-C287 T. rubrum PF-168 PF-278 PF-283PF-307 PF-527 PF-531 PF-547 PF-672 PF-C019 PF-C021 PF-C252 PF-S028 M.furfur PF-S028

Conversely, the activity against a particular microorganism or group ofmicroorganisms can be increased by increasing the number of peptides orpeptide domains with activity against that microorganism or group ofmicroorganisms.

Thus, for example, in certain embodiments, a peptide or compositioneffective to kill or inhibit the growth or proliferation of a yeast orfungus can comprise or more peptides and/or one or more peptide domainshaving sequences selected from the group consisting of PF-148, PF-168,PF-448, PF-525, PF-527, PF-529, PF-531, PF-545, PF-672, PF-0019, PF-278,PF-307, PF-672, PF-C021, PF-C157, PF-C220, PF-C252, PF-C287, PF-S028,PF-168, PF-278, PF-283, PF-307, PF-527, PF-531, PF-547, PF-672, PF-0019,PF-C021, PF-C252, and PF-S028. A peptide or composition effective tokill or inhibit the growth or proliferation of Aspergillus niger cancomprise one or more peptides and/or one or more peptide domains havingsequences selected from the group consisting of PF-148, PF-168, PF-448,PF-525, PF-527, PF-529, PF-531, PF-545, PF-672, and PF-0019. A peptideor composition effective to kill or inhibit the growth or proliferationof Candida albicans can comprise one or more peptides and/or one or morepeptide domains having sequences selected from the group consisting ofPF-278, PF-307, PF-672, PF-C021, PF-C157, PF-C220, PF-C252, and PF-C287.A peptide or composition effective to kill or inhibit the growth orproliferation of Malassezia furfur can comprise a peptides and/or apeptide domains having sequence of PF-S028. A peptide or compositioneffective to kill or inhibit the growth or proliferation of Trichophytonrubrum can comprise one or more peptides and/or one or more peptidedomains having sequences selected from the group consisting of PF-168,PF-278, PF-283, PF-307, PF-527, PF-531, PF-547, PF-672, PF-0019,PF-C021, PF-C252, PF-S028.

In certain embodiments a peptide or composition effective to kill orinhibit the growth or proliferation of a bacterium can comprise one ormore peptides and/or one or more peptide domains having sequencesselected from the group consisting of PF-006, PF-530, PF-531, PF-538,PF-C163, PF-C239, PF-C252, PF-C287, PF-006, PF-148, PF-283, PF-307,PF-322, PF-497, PF-499, PF-527, PF-531, PF-545, PF-547, PF-672, PF-S028,PF-522, PF-531, PF-538, PF-600, PF-606, PF-672, PF-C239, PF-C252,PF-006, PF-168, PF-209, PF-527, PF-545, PF-583, PF-606, PF-672, PF-C252,PF-S028, PF-S028, PF-C252, PF-531, PF-527, PF-S028, PF-C163, PF-C239,PF-C252, PF-C287, PF-278, PF-283, PF-527, PF-531, PF-583, PF-606,PF-672, PF-C163, PF-C252, PF-S028, PF-531, PF-547, PF-601, PF-0019,PF-C239, PF-C252, PF-S028, PF-437, PF-448, PF-511, PF-512, PF-520,PF-521, PF-523, PF-524, PF-525, PF-529, PF-600, and PF-606.

In certain embodiments a peptide or composition effective to kill orinhibit the growth or proliferation of a gram positive bacterium cancomprise one or more peptides and/or one or more peptide domains havingsequences selected from the group consisting of PF-006, PF-148, PF-168,PF-209, PF-278, PF-283, PF-307, PF-322, PF-437, PF-448, PF-497, PF-499,PF-511, PF-512, PF-520, PF-521, PF-522, PF-523, PF-524, PF-525, PF-527,PF-529, PF-531, PF-538, PF-545, PF-547, PF-583, PF-600, PF-601, PF-606,PF-672, PF-C019, PF-C163, PF-C239, PF-C252, PF-C287, PF-S028. A peptideor composition effective to kill or inhibit the growth or proliferationof Actinomyces naeslundii can comprise one or more peptides and/or oneor more peptide domains having sequences selected from the groupconsisting of PF-C163, PF-C239, PF-C252, and PF-C287. A peptide orcomposition effective to kill or inhibit the growth or proliferationBacillus subtilis can comprise one or more peptides and/or one or morepeptide domains having sequences selected from the group consisting ofPF-006, PF-148, PF-283, PF-307, PF-322, PF-497, PF-499, PF-527, PF-531,PF-545, PF-547, PF-672, and PF-S028. A peptide or composition effectiveto kill or inhibit the growth or proliferation of Clostridium difficilecan comprise one or more peptides and/or one or more peptide domainshaving sequences selected from the group consisting of PF-522, PF-531,and PF-538. A peptide or composition effective to kill or inhibit thegrowth or proliferation of Enterococcus faecalis can comprise a peptideand/or one or more peptide domain having the amino acid sequence ofPF-672. A peptide or composition effective to kill or inhibit the growthor proliferation of Methicillin-resistant Staphylococcus aureus (MRSA)can comprise one or more peptides and/or one or more peptide domainshaving sequences selected from the group consisting of PF-006, PF-168,PF-209, PF-527, PF-545, PF-583, PF-606, PF-672, PF-C252, and PF-S028. Apeptide or composition effective to kill or inhibit the growth orproliferation of S. epidermidis can comprise one or more peptides and/orone or more peptide domains having sequences selected from the groupconsisting of PF-278, PF-283, PF-527, PF-531, PF-583, PF-606, PF-672,PF-C163, PF-C252, and PF-S028. A peptide or composition effective tokill or inhibit the growth or proliferation of Streptococcus cancomprise one or more peptides and/or one or more peptide domains havingsequences selected from the group consisting of PF-531, PF-547, PF-601,PF-0019, PF-C239, PF-C252, and PF-S028. A peptide or compositioneffective to kill or inhibit the growth or proliferation ofStreptococcus pneumoniae can comprise one or more peptides and/or one ormore peptide domains having sequences selected from the group consistingof PF-437, PF-448, PF-511, PF-512, PF-520, PF-521, PF-523, PF-524,PF-525, PF-529, PF-600, and PF-606.

In certain embodiments a peptide or composition effective to kill orinhibit the growth or proliferation of effective to kill or inhibit thegrowth or proliferation of a gram negative bacterium can comprise one ormore peptides and/or one or more peptide domains having sequencesselected from the group consisting of PF-006, PF-527, PF-530, PF-531,PF-538, PF-600, PF-606, PF-C163, PF-C239, PF-C252, PF-C287, and PF-S028.A peptide or composition effective to kill or inhibit the growth orproliferation of Acinetobacter baumannii can comprise one or morepeptides and/or one or more peptide domains having sequences selectedfrom the group consisting of PF-006, PF-530, PF-531, and PF-538. Apeptide or composition effective to kill or inhibit the growth orproliferation of Escherichia coli can comprise one or more peptidesand/or one or more peptide domains having sequences selected from thegroup consisting of PF-600, and PF-606. A peptide or compositioneffective to kill or inhibit the growth or proliferation ofFusobacterium nucleatum can comprise one or more peptides and/or one ormore peptide domains having sequences selected from the group consistingof PF-C239, and PF-C252. A peptide or composition effective to kill orinhibit the growth or proliferation of Pseudomonas aeruginosa cancomprise one or more peptides and/or one or more peptide domains havingsequences selected from the group consisting of PF-S028, PF-C252,PF-531, and PF-527. A peptide or composition effective to kill orinhibit the growth or proliferation of Porphyromonas gingivalis cancomprise one or more peptides and/or one or more peptide domains havingsequences selected from the group consisting of PF-S028, PF-C163,PF-C239, PF-C252, and PF-C287.

A peptide or composition effective to kill or inhibit the growth orproliferation of Corynebacterium jeikium can comprise one or morepeptides and/or one or more peptide domains having sequences selectedfrom the group consisting of PF-531, PF-S028, PF-527.

II. Chimeric Constructs.

In various embodiments this invention provides chimeric moietiescomprising antimicrobial peptides attached to targeting moieties, todetectable labels, and/or to opsonins, lysosomes, or otherinternalization signals.

Targeted Antimicrobial Peptides.

In certain embodiments the antimicrobial peptides (e.g., peptidescomprising one or more amino acid sequences found in Table 3) can beattached (directly or through a linker) to one or more targetingmoieties to specifically or preferentially deliver the AMP construct toa target microorganism, to a particular cell or tissue, and the like. Invarious embodiments the targeting moieties preferentially and/orspecifically bind to a microorganism (e.g., a bacterium, a fungus, ayeast, etc.).

Targeting Peptides.

In certain embodiments the targeting moieties include, but are notlimited to peptides that preferentially bind particular microorganisms(e.g., bacteria, fungi, yeasts, protozoa, algae, viruses, etc.) orgroups of such microorganisms, antibodies that bind particularmicroorganisms or groups of microorganisms, receptor ligands that bindparticular microorganisms or groups of microorganisms, porphyrins (e.g.,metalloporphyrins), lectins that bind particular microorganisms orgroups of microorganisms, and the like. As indicated it will beappreciated that references to microorganisms or groups of microorganismcan include bacteria or groups of bacteria, viruses or groups ofviruses, yeasts or groups of yeasts, protozoa or groups of protozoa,viruses or groups of viruses, and the like.

Suitable targeting peptides are disclosed, for example in US PatentPublication No: 2008-0170991 (WO/2008/030988) and include for example,C16 (TFFRLFNRSFTQALGK, SEQ ID NO:61), M8 (TFFRLFNR, SEQ ID NO:62), 1903(NIFEYFLE, SEQ ID NO:63) as well as SEQ ID NOs:34-35 and 54-97 in thatpublication. Additionally, 1T-6 (KFINGVLSQFVLERK, SEQ ID NO:64), 1T-18(YSKTLHFAD, SEQ ID NO:65), 1T-30 (GKAKPYQVRQVLRAVDKLETRRKKGGR, SEQ IDNO:66), PF-S024 (SKRGRKRKDRRKKKANHGKRPNS, SEQ ID NO:67) are can aresuitable targeting peptides/domains. Other suitable targeting peptidesare disclosed, for example, in priority documents U.S. Ser. No.61/142,830, filed Jan. 6, 2009, U.S. Ser. No. 61/151,445, filed Feb. 10,2009, U.S. Ser. No. 61/243,905, filed Sep. 18, 2009, and U.S. Ser. No.61/243,930, filed Sep. 18, 2009.

In certain embodiments, targeting peptides consist of or comprise one ormore of the C. albicans binding sequences shown in Table 6, or retro,inverse, retroinverso, or β forms thereof

TABLE 6  Illustrative list of peptides that bind to Candida albicans.SEQ ID Target(s) Targeting Peptide Sequence ID No. PF-060 hyphae HSSHL68 PF-024 hyphae DLRKAK 69 PF-636 hyphae LVRLA 70 PF-178 hyphaeEVYSSPTNNVAITVQNN 71 PF-761 hyphae SKFELVNYASGCSCGADCKCASETECKCAS 72 KKPF-770 hyphae GVGIGFIMMGVVGYAVKLVHIPIRYLIV 73 1T-65 hyphaeHARAAVGVAELPRGAAVEVELIAAVRP 74 PF-141 hyphae VVRRFQGM 75 PF-543 hyphaeNILFGIIGFVVAMTAAVIVTAISIAK 76 PF-634 hyphaeMPKARPVNHNKKKSKITIKSNFTLFYMFNP 77 PF-040 hyphaeMIHLTKQNTMEALHFIKQFYDMFFILNFNV 78 PF-051 hyphaeRFFNFEIKKSTKVDYVFAHVDLSDV 79 PF-580 hyphaeEILNNNQVIKELTMKYKTQFESNLGGWTAR 80 ARR PF-583 hyphaeKFQGEFTNIGQSYIVSASHMSTSLNTGK 81 1T-36 hyphae VYRHLRFIDGKLVEIRLERK 82PF-206 hyphae KLRSASKKSLQEKSCGIMPEKPAG 83 1T-13 yeast andFRSPCINNNSLQPPGVYPAR 84 hyphal forms  1T-21 yeast andYVEEAVRAALKKEARISTEDTPVNLPSFDC 85 hyphal forms PF-030 yeast andMTCHQAPTTTHQSNMA 86 hyphal forms  PF-463 yeast andMVILVFSLIFIFTDNYLVYQSKSIKEDVMI 87 hyphal forms  PF-380 yeast andKKIIPLITLFVVTLVG 88 hyphal forms  PF-515 yeast andDKSTQDKDIKQAKLLAQELGL-NH2 89 hyphal forms PF-458 yeast andISLIIFIMLFVVALFKCITNYKHQS 90 hyphal forms PF-S018 yeast and GMPQIPRLRI91 hyphal forms 1T-16 yeast and IDMR 92 hyphal forms PF-211 yeast andDSFDSLSPFRERGGEREDGCDAMPLP 93 hyphal forms PF-002 yeast and NDDAQ 94hyphal forms  PF-S003 yeast and ALALLKQDLLNFEGRGRIITSTYLQFNEGCV 95hyphal forms P PF-021 yeast and FSLNFSKQKYVTVN 96 hyphal forms 1T-14yeast and ALAGLAGLISGK 97 hyphal forms  1T-15 yeast and DVILRVEAQ 98hyphal forms PF-629 yeast and GLAAIATVFALY 99 hyphal forms PF-617yeast and PMNAAEPE 100 hyphal forms  PF-621 yeast and PPSSFLV 101hyphal forms PF-631 yeast and LIIYFSKTGNTARATRQI 102 hyphal forms PF-009 yeast and SKKYNHILNQENR 103 hyphal forms  1T-17 yeast andNNAIVYIS 104 hyphal forms  1T-20 yeast and PALVDLSNKEAVWAVLDDHS 105hyphal forms 1T-68 yeast cells GGTKEIVYQRG 106 1T-70 yeast cellsNRQAQGERAHGEQQG 107 PF-167 yeast cells AIEGVIKKGACFKLLRHEMF 108 PF-168yeast cells VLPFPAIPLSRRRACVAAPRPRSRQRAS 109 PF-170 yeast cellsRLARGRPTNLCGRRG 110 PF-176 yeast cells RLTSNQFLTRITPFVFAQH 111

The peptides in Table 6 were tested for binding efficiency at 3.13,6.25, 12.5 and 25.0 μM concentrations in 1× phosphate-buffered saline or20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 150 mMsodium chloride, 1 mM magnesium chloride and 0.1% CTAB.

Targeting Antibodies.

In certain embodiments the targeting moieties can comprise one or moreantibodies that bind specifically or preferentially a microorganism orgroup of microorganisms (e.g., bacteria, fungi, yeasts, protozoa,viruses, algae, etc.). The antibodies are selected to bind an epitopecharacteristic or the particular target microorganism(s). In variousembodiments such epitopes or antigens are typically is gram-positive orgram-negative specific, or genus-specific, or species-specific, orstrain specific and located on the surface of a target microbialorganism. The antibody that binds the epitope or antigen can direct ananti-microbial peptide moiety to the site. Furthermore, in certainembodiments the antibody itself can provide anti-microbial activity inaddition to the activity provided by the AMP since the antibody mayengage an immune system effector (e.g., a T-cell) and thereby elicit anantibody-associated immune response, e.g., a humoral immune response.

Antibodies that bind particular target microorganisms can be made usingany methods readily available to one skilled in the art. For example, asdescribed in U.S. Pat. No. 6,231,857 (incorporated herein by reference)three monoclonal antibodies, i.e., SWLA1, SWLA2, and SWLA3 have beenmade against S. mutans. Monoclonal antibodies obtained from non-humananimals to be used in a targeting moiety can also be humanized by anymeans available in the art to decrease their immunogenicity and increasetheir ability to elicit anti-microbial immune response of a human

Illustrative antibodies that bind various microorganisms are shown inTable 7.

TABLE 7 Illustrative antibodies that bind target microorganisms. SourceAntibody U.S. Pat. No. 7,195,763 Polyclonal/monoclonal binds specificGram(+) cell wall repeats U.S. Pat. No. 6,939,543 Antibodies againstG(+) LTA U.S. Pat. No. 7,169,903 Antibodies against G(+) peptidoglycanU.S. Pat. No. 6,231,857 Antibody against S. mutans (Shi) U.S. Pat. No.5,484,591 Gram(−) binding antibodies US 2007/0231321 Diabody binding toStreptococcus surface antigen I/II US 2003/0124635 Antibody against S.mutans US 2006/0127372 Antibodies to Actinomyces naeslundii,Lactobacillus casei US 2003/0092086 Antibody to S. sobrinus U.S. Pat.No. 7,364,738 Monoclonal antibodies to the ClfA protein in S. aureusU.S. Pat. No. 7,632,502 Antibodies against C. albicans U.S. Pat. No.7,608,265 Monoclonal against C. difficile U.S. Pat. No. 4,777,136Monoclonal Antibodies against Pseudomonas aeruginosa see, e.g., ab20429,ab20560, Antibody against S. pneumoniae ab79522, ab35165, ab65602 fromAbCAMm Cambridge Science Park, U.K.

The targeting moiety can be attached directly to the AMP or compound AMPconstruct or it can be attached via a linker (e.g., as shown in Table4).

Amps Attached to Detectable Labels.

In certain embodiments chimeric moieties are provided comprising theantimicrobial peptides (e.g., peptides comprising one or more amino acidsequences found in Table 3) attached directly or through a linker to adetectable label. Such chimeric moieties are effective for detecting thepresence and/or quantity, and/or location of the microorganism(s) thatmay be bound by the AMP(s). Similarly these chimeric moieties are usefulto identify cells and/or tissues and/or food stuffs and/or othercompositions that are infected with the targeted microorganism(s).

Detectable labels suitable for use in such chimeric moieties include anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical, or chemical means. Illustrativeuseful labels include, but are not limited to, biotin for staining withlabeled streptavidin conjugates, avidin or streptavidin for labelingwith biotin conjugates fluorescent dyes (e.g., fluorescein, texas red,rhodamine, green fluorescent protein, and the like, see, e.g., MolecularProbes, Eugene, Oreg., USA), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P,⁹⁹Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu, 641Cu,⁵²Fe, ^(52m)Mn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn,¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm,¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, ¹¹¹Ag,and the like), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), various colorimetriclabels, magnetic or paramagnetic labels (e.g., magnetic and/orparamagnetic nanoparticles), spin labels, radio-opaque labels, and thelike. Patents teaching the use of such labels include, for example, U.S.Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241.

It will be recognized that fluorescent labels are not to be limited tosingle species organic molecules, but include inorganic molecules,multi-molecular mixtures of organic and/or inorganic molecules,crystals, heteropolymers, and the like. Thus, for example, CdSe—CdScore-shell nanocrystals enclosed in a silica shell can be easilyderivatized for coupling to a biological molecule (Bruchez et al. (1998)Science, 281: 2013-2016). Similarly, highly fluorescent quantum dots(zinc sulfide-capped cadmium selenide) have been covalently coupled tobiomolecules for use in ultrasensitive biological detection (Warren andNie (1998) Science, 281: 2016-2018).

In various embodiments spin labels are provided by reporter moleculeswith an unpaired electron spin which can be detected by electron spinresonance (ESR) spectroscopy. Illustrative spin labels include organicfree radicals, transitional metal complexes, particularly vanadium,copper, iron, and manganese, and the like. Exemplary spin labelsinclude, for example, nitroxide free radicals.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence, e.g., by microscopy,visual inspection, via photographic film, by the use of electronicdetectors such as charge coupled devices (CCDs) or photomultipliers andthe like. Similarly, enzymatic labels may be detected by providingappropriate substrates for the enzyme and detecting the resultingreaction product. Finally, simple colorimetric labels may be detectedsimply by observing the color associated with the label.

AMPs Attached to Targeting Enhancers/Opsonins

In certain embodiments compositions are contemplated that incorporate atargeting enhancer (e.g., an opsonin) attached to the AMP and/or to atargeted AMP. Targeting enhancers include moieties that increase bindingaffinity, and/or binding specificity, and/or internalization of a moietyby the target cell/microorganism.

Accordingly, in certain embodiments, an AMP and/or a targetedantimicrobial molecule is attached (e.g., conjugated) to an opsonin.When bound to a target cell through the targeting peptide, the opsonincomponent encourages phagocytosis and destruction by residentmacrophages, dendritic cells, monocytes, or PMNs. Opsonins contemplatedfor conjugation can be of a direct or indirect type.

Direct opsonins include, fore example, any bacterial surface antigen,PAMP (pathogen-associated molecular pattern), or other moleculerecognized by host PRRs (pathogen recognizing receptors). Opsonins caninclude, but are not limited to, bacterial protein, lipid, nucleic acid,charbohydrate and/or oligosaccharide moieties.

In certain embodiments opsonins include, but are not limited to,N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-galactosamine (GlaNAc),N-acetylglucosamine-containing muramyl peptides, NAG-muramyl peptides,NAG-NAM, peptidoglycan, teichoic acid, lipoteichoic acid, LPS,o-antigen, mannose, fucose, ManNAc, galactose, maltose, glucose,glucosamine, sucrose, mannosamine,galactose-alpha-1,3-galactosyl-beta-1,4-N-acetyl glucosamine, oralpha-1,3-gal-gal, or other sugars.

In certain embodiments, opsonins include indirect opsonins Indirectopsonins function through binding to a direct opsonin already present.For example an Fc portion of an antibody, a sugar-binding lectin protein(example MBL), or host complement factors (example C3b, C4b, iC3b).

In certain embodiments the opsonin is togalactose-alpha-1,3-galactosyl-beta-1,4-N-acetyl glucosamine, oralpha-1,3-gal-gal.

Other examples of opsonin molecules include, but are not limited toantibodies (e.g., IgG and IgA), components of the complement system(e.g., C3b, C4b, and iC3b), mannose-binding lectin (MBL) (initiates theformation of C3b), and the like.

Methods of coupling an opsonin to a targeting moiety are well known tothose of skill in the art (see, e.g., discussion below regardingattachment of effectors to targeting moieties).

III. Protecting Groups.

While the various peptides (e.g., targeted peptides, antimicrobialpeptides, compound AMPs, etc.) described herein may be illustrated withno protecting groups, in certain embodiments they can bear one, two,three, four, or more protecting groups. In various embodiments, theprotecting groups can be coupled to the C- and/or N-terminus of thepeptide(s) and/or to one or more internal residues comprising thepeptide(s) (e.g., one or more R-groups on the constituent amino acidscan be blocked). Thus, for example, in certain embodiments, any of thepeptides described herein can bear, e.g., an acetyl group protecting theamino terminus and/or an amide group protecting the carboxyl terminus.Illustrative examples of such a protected peptides include, but are notlimited to: YIQFHLNQQPRPKVKKIKIFL-NH₂ (SEQ ID NO:3),WSRVPGHSDTGWKVWHRW-NH₂ (SEQ ID NO:8), RESKLIAMADMIRRRI-NH₂ (SEQ IDNO:11), LSEMERRRLRKRA-NH₂ (SEQ ID NO:4), FELVDWLETNLGKILKSKSA-NH₂ (SEQID NO:27), and LGLTAGVAYAAQPTNQPTNQPTNQPTNQPTNQPTNQPRW-NH₂ (SEQ IDNO:33). Of course, the —NH2 protecting group can be can be eliminatedand/or substituted with another protecting group as described herein.

Without being bound by a particular theory, it was a discovered thataddition of a protecting group, particularly to the carboxyl terminus,and/or in certain embodiments to the amino terminus, can improve thestability and efficacy of the peptide.

A wide number of protecting groups are suitable for this purpose. Suchgroups include, but are not limited to acetyl, amide, and alkyl groupswith acetyl and alkyl groups being particularly preferred for N-terminalprotection and amide groups being preferred for carboxyl terminalprotection. In certain particularly preferred embodiments, theprotecting groups include, but are not limited to alkyl chains as infatty acids, propionyl, formyl, and others. Particularly preferredcarboxyl protecting groups include amides, esters, and ether-formingprotecting groups. In one preferred embodiment, an acetyl group is usedto protect the amino terminus and an amide group is used to protect thecarboxyl terminus. These blocking groups enhance the helix-formingtendencies of the peptides. Certain particularly preferred blockinggroups include alkyl groups of various lengths, e.g., groups having theformula: CH₃—(CH₂)_(n)—CO— where n ranges from about 1 to about 20,preferably from about 1 to about 16 or 18, more preferably from about 3to about 13, and most preferably from about 3 to about 10.

In certain embodiments, the acid group on the C-terminal can be blockedwith an alcohol, aldehyde or ketone group and/or the N-terminal residuecan have the natural amide group, or be blocked with an acyl, carboxylicacid, alcohol, aldehyde, or ketone group.

In certain embodiments, the protecting groups include, but are notlimited to alkyl chains as in fatty acids, propionyl, formyl, andothers. Particularly preferred carboxyl protecting groups includeamides, esters, and ether-forming protecting groups. In one embodiment,an acetyl group is used to protect the amino terminus and/or an aminogroup is used to protect the carboxyl terminus (i.e., amidated carboxylterminus). In certain embodiments blocking groups include alkyl groupsof various lengths, e.g., groups having the formula: CH₃—(CH₂)_(n)—CO—where n ranges from about 3 to about 20, preferably from about 3 toabout 16, more preferably from about 3 to about 13, and most preferablyfrom about 3 to about 10.

Other protecting groups include, but are not limited to Fmoc,t-butoxycarbonyl (t-BOC), 9-fluoreneacetyl group, 1-fluorenecarboxylicgroup, 9-florenecarboxylic group, 9-fluorenone-1-carboxylic group,benzyloxycarbonyl, xanthyl (Xan), trityl (Trt), 4-methyltrityl (Mtt),4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), tosyl(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl(MeBzl), 4-methoxybenzyl (MeOBzl), benzyloxy (BzlO), benzyl (Bzl),benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl—Z),2-bromobenzyloxycarbonyl (2-Br—Z), benzyloxymethyl (Bom), cyclohexyloxy(cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), acetyl(Ac), and trifluoroacetyl (TFA).

Protecting/blocking groups are well known to those of skill as aremethods of coupling such groups to the appropriate residue(s) comprisingthe peptides of this invention (see, e.g., Greene et al., (1991)Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc.Somerset, N.J.). In illustrative embodiment, for example, acetylation isaccomplished during the synthesis when the peptide is on the resin usingacetic anhydride. Amide protection can be achieved by the selection of aproper resin for the synthesis. For example, a rink amide resin can beused. After the completion of the synthesis, the semipermanentprotecting groups on acidic bifunctional amino acids such as Asp and Gluand basic amino acid Lys, hydroxyl of Tyr are all simultaneouslyremoved. The peptides released from such a resin using acidic treatmentcomes out with the n-terminal protected as acetyl and the carboxylprotected as NH₂ and with the simultaneous removal of all of the otherprotecting groups.

Where amino acid sequences are disclosed herein, amino acid sequencescomprising, one or more protecting groups, e.g., as described above (orany other commercially available protecting groups for amino acids used,e.g., in boc or fmoc peptide synthesis) are also contemplated.

IV. Peptide Preparation.

The peptides described herein can be chemically synthesized usingstandard chemical peptide synthesis techniques or, particularly wherethe peptide does not comprise “D” amino acid residues, the peptide canbe recombinantly expressed. Where the “D” polypeptides are recombinantlyexpressed, a host organism (e.g. bacteria, plant, fungal cells, etc.)can be cultured in an environment where one or more of the amino acidsis provided to the organism exclusively in a D form. Recombinantlyexpressed peptides in such a system then incorporate those D aminoacids.

In certain embodiments, D amino acids can be incorporated inrecombinantly expressed peptides using modified amino acyl-tRNAsynthetases that recognize D-amino acids.

Chemical Synthesis.

In certain embodiments the peptides are chemically synthesized by any ofa number of fluid or solid phase peptide synthesis techniques known tothose of skill in the art. Solid phase synthesis in which the C-terminalamino acid of the sequence is attached to an insoluble support followedby sequential addition of the remaining amino acids in the sequence is apreferred method for the chemical synthesis of the polypeptides of thisinvention. Techniques for solid phase synthesis are well known to thoseof skill in the art and are described, for example, by Barany andMerrifield (1963) Solid-Phase Peptide Synthesis; pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield et al. (1963) J. Am. Chem. Soc.,85: 2149-2156, and Stewart et al. (1984) Solid Phase Peptide Synthesis,2nd ed. Pierce Chem. Co., Rockford, Ill.

In one illustrative embodiment, the peptides can be synthesized by thesolid phase peptide synthesis procedure using a benzhyderylamine resin(Beckman Bioproducts, 0.59 mmol of NH₂/g of resin) as the solid support.The COOH terminal amino acid (e.g., t-butylcarbonyl-Phe) is attached tothe solid support through a 4-(oxymethyl)phenacetyl group. This is amore stable linkage than the conventional benzyl ester linkage, yet thefinished peptide can still be cleaved by hydrogenation. Transferhydrogenation using formic acid as the hydrogen donor can be used forthis purpose.

It is noted that in the chemical synthesis of peptides, particularlypeptides comprising D amino acids, the synthesis usually produces anumber of truncated peptides in addition to the desired full-lengthproduct. Thus, the peptides are typically purified using, e.g., HPLC.

D-amino acids, beta amino acids, non-natural amino acids, and the likecan be incorporated at one or more positions in the peptide simply byusing the appropriately derivatized amino acid residue in the chemicalsynthesis. Modified residues for solid phase peptide synthesis arecommercially available from a number of suppliers (see, e.g., AdvancedChem Tech, Louisville; Nova Biochem, San Diego; Sigma, St Louis; BachemCalifornia Inc., Torrance, etc.). The D-form and/or otherwise modifiedamino acids can be completely omitted or incorporated at any position inthe peptide as desired. Thus, for example, in certain embodiments, thepeptide can comprise a single modified acid, while in other embodiments,the peptide comprises at least two, generally at least three, moregenerally at least four, most generally at least five, preferably atleast six, more preferably at least seven or even all modified aminoacids. In certain embodiments, essentially every amino acid is a D-formamino acid.

Recombinant Expression.

As indicated above, the antimicrobial peptides can also be recombinantlyexpressed. Accordingly, in certain embodiments, the antimicrobialpeptides and/or targeting moieties, and/or fusion proteins of thisinvention are synthesized using recombinant expression systems.Generally this involves creating a DNA sequence that encodes the desiredpeptide or fusion protein, placing the DNA in an expression cassetteunder the control of a particular promoter, expressing the peptide orfusion protein in a host, isolating the expressed peptide or fusionprotein and, if required, renaturing the peptide or fusion protein.

DNA encoding the peptide(s) or fusion protein(s) described herein can beprepared by any suitable method as described above, including, forexample, cloning and restriction of appropriate sequences or directchemical synthesis.

This nucleic acid can be easily ligated into an appropriate vectorcontaining appropriate expression control sequences (e.g. promoter,enhancer, etc.), and, optionally, containing one or more selectablemarkers (e.g. antibiotic resistance genes).

The nucleic acid sequences encoding the peptides or fusion proteinsdescribed herein can be expressed in a variety of host cells, including,but not limited to, E. coli, other bacterial hosts, yeast, fungus, andvarious higher eukaryotic cells such as insect cells (e.g. SF3), theCOS, CHO and HeLa cells lines and myeloma cell lines. The recombinantprotein gene will typically be operably linked to appropriate expressioncontrol sequences for each host. For E. coli this can include a promotersuch as the T7, tip, or lambda promoters, a ribosome binding site andpreferably a transcription termination signal. For eukaryotic cells, thecontrol sequences can include a promoter and often an enhancer (e.g., anenhancer derived from immunoglobulin genes, SV40, cytomegalovirus,etc.), and a polyadenylation sequence, and may include splice donor andacceptor sequences.

The plasmids can be transferred into the chosen host cell by well-knownmethods such as calcium chloride transformation for E. coli and calciumphosphate treatment or electroporation for mammalian cells. Cellstransformed by the plasmids can be selected by resistance to antibioticsconferred by genes contained on the plasmids, such as amp, gpt, neo,hyg, and the like.

Once expressed, the recombinant peptide(s) or fusion protein(s) can bepurified according to standard procedures of the art, including ammoniumsulfate precipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, (1982) ProteinPurification, Springer-Verlag, N.Y.; Deutscher (1990) Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y.). Substantially pure compositions of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity are mostpreferred.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the peptide(s) or fusionprotein(s) may possess a conformation substantially different thandesired native conformation. In this case, it may be necessary todenature and reduce the peptide or fusion protein and then to cause themolecule to re-fold into the preferred conformation. Methods of reducingand denaturing proteins and inducing re-folding are well known to thoseof skill in the art (see, e.g., Debinski et al. (1993) J. Biol. Chem.,268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4:581-585; and Buchner, et al., (1992) Anal. Biochem., 205: 263-270).Debinski et al., for example, describes the denaturation and reductionof inclusion body proteins in guanidine-DTE. The protein is thenrefolded in a redox buffer containing oxidized glutathione andL-arginine.

One of skill would recognize that modifications can be made to thepeptide(s) and/or fusion protein(s) proteins without diminishing theirbiological activity. Some modifications may be made to facilitate thecloning, expression, or incorporation of the targeting molecule into afusion protein. Such modifications are well known to those of skill inthe art and include, for example, a methionine added at the aminoterminus to provide an initiation site, or additional amino acids (e.g.,poly His) placed on either terminus to create conveniently locatedrestriction sites or termination codons or purification sequences.

Attachment of AMPS to Each Other and/or to a Targeting Moiety,Detectable Label and/or Opsonin.

Chemical Conjugation.

Chimeric moieties are formed by joining one or more of the AMPs moietiesdescribed herein to each other and/or to one or more targeting moietiesand/or to detectable labels and/or to opsonins. In certain embodimentsthe components are attached directly to each other via naturallyoccurring reactive groups or the AMPs and/or targeting moieties can befunctionalized to provide such reactive groups.

In various embodiments the AMPs are attached to each other and/or totargeting moieties via one or more linking agents. Thus, in variousembodiments the AMPs and/or targeting moieties can be conjugated via asingle linking agent or multiple linking agents. For example, they canbe conjugated via a single multifunctional (e.g., bi-, tri-, or tetra-)linking agent or a pair of complementary linking agents. In anotherembodiment, the AMPs and/or targeting moieties are conjugated via two,three, or more linking agents. Suitable linking agents include, but arenot limited to, e.g., functional groups, affinity agents, stabilizinggroups, and combinations thereof.

In certain embodiments the linking agent is or comprises a functionalgroup. Functional groups include monofunctional linkers comprising areactive group as well as multifunctional crosslinkers comprising two ormore reactive groups capable of forming a bond with two or moredifferent functional targets (e.g., labels, proteins, macromolecules,semiconductor nanocrystals, or substrate). In some preferredembodiments, the multifunctional crosslinkers are heterobifunctionalcrosslinkers comprising two or more different reactive groups.

Suitable reactive groups include, but are not limited to thiol (—SH),carboxylate (COOH), carboxyl (—COOH), carbonyl, amine (NH₂), hydroxyl(—OH), aldehyde (—CHO), alcohol (ROH), ketone (R₂CO), active hydrogen,ester, sulfhydryl (SH), phosphate (—PO₃), or photoreactive moieties.Amine reactive groups include, but are not limited to e.g.,isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonylchlorides, aldehydes and glyoxals, epoxides and oxiranes, carbonates,arylating agents, imidoesters, carbodiimides, and anhydrides.Thiol-reactive groups include, but are not limited to e.g., haloacetyland alkyl halide derivates, maleimides, aziridines, acryloylderivatives, arylating agents, and thiol disulfides exchange reagents.Carboxylate reactive groups include, but are not limited to e.g.,diazoalkanes and diazoacetyl compounds, such as carbonyldiimidazoles andcarbodiimides. Hydroxyl reactive groups include, but are not limited toe.g., epoxides and oxiranes, carbonyldiimidazole, oxidation withperiodate, N,N′-disuccinimidyl carbonate or N-hydroxylsuccimidylchloroformate, enzymatic oxidation, alkyl halogens, and isocyanates.Aldehyde and ketone reactive groups include, but are not limited toe.g., hydrazine derivatives for schiff base formation or reductionamination. Active hydrogen reactive groups include, but are not limitedto e.g., diazonium derivatives for mannich condensation and iodinationreactions. Photoreactive groups include, but are not limited to e.g.,aryl azides and halogenated aryl azides, benzophenones, diazo compounds,and diazirine derivatives.

Other suitable reactive groups and classes of reactions useful informing chimeric moieties include those that are well known in the artof bioconjugate chemistry. Currently favored classes of reactionsavailable with reactive chelates are those which proceed underrelatively mild conditions. These include, but are not limited to,nucleophilic substitutions (e.g., reactions of amines and alcohols withacyl halides, active esters), electrophilic substitutions (e.g., enaminereactions), and additions to carbon-carbon and carbon-heteroatommultiple bonds (e.g., Michael reaction, Diels-Alder addition). These andother useful reactions are discussed in, for example, March (1985)Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York,Hermanson (1996) Bioconjugate Techniques, Academic Press, San Diego; andFeeney et al. (1982) Modification of Proteins; Advances in ChemistrySeries, Vol. 198, American Chemical Society, Washington, D.C.

A “linker” or “linking agent” as used herein, is a molecule that is usedto join two or more molecules. In certain embodiments the linker istypically capable of forming covalent bonds to both molecule(s) (e.g.,the targeting moiety and the effector). Suitable linkers are well knownto those of skill in the art and include, but are not limited to,straight or branched-chain carbon linkers, heterocyclic carbon linkers,or peptide linkers. In certain embodiments the linkers can be joined tothe constituent amino acids through their side groups (e.g., through adisulfide linkage to cysteine). However, in certain embodiments, thelinkers will be joined to the alpha carbon amino and carboxyl groups ofthe terminal amino acids. An list of suitable linkers is shown in Table4.

A bifunctional linker having one functional group reactive with a groupon one molecule (e.g., a targeting peptide), and another group reactiveon the other molecule (e.g., an antimicrobial peptide), can be used toform the desired conjugate. Alternatively, derivatization can beperformed to provide functional groups. Thus, for example, proceduresfor the generation of free sulfhydryl groups on peptides are also known(See U.S. Pat. No. 4,659,839).

In certain embodiments the linking agent is a heterobifunctionalcrosslinker comprising two or more different reactive groups that form aheterocyclic ring that can interact with a peptide. For example, aheterobifunctional crosslinker such as cysteine may comprise an aminereactive group and a thiol-reactive group can interact with an aldehydeon a derivatized peptide. Additional combinations of reactive groupssuitable for heterobifunctional crosslinkers include, for example,amine- and sulfhydryl reactive groups; carbonyl and sulfhydryl reactivegroups; amine and photoreactive groups; sulfhydryl and photoreactivegroups; carbonyl and photoreactive groups; carboxylate and photoreactivegroups; and arginine and photoreactive groups. In one embodiment, theheterobifunctional crosslinker is SMCC.

Many procedures and linker molecules for attachment of various moleculesto peptides or proteins are known (see, e.g., European PatentApplication No. 188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148,4,699,784; 4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al.(1987) Cancer Res. 47: 4071-4075). Illustrative linking protocols areprovided herein in Examples 2 and 3.

Fusion Proteins.

In certain embodiments where the moieties to be joined (AMPs and/ortargeting moieties) are all peptides, the chimeric moiety can bechemically synthesized or recombinantly expressed as a fusion protein(i.e., a chimeric fusion protein).

In certain embodiments the chimeric fusion proteins are synthesizedusing recombinant DNA methodology. Generally this involves creating aDNA sequence that encodes the fusion protein, placing the DNA in anexpression cassette under the control of a particular promoter,expressing the protein in a host, isolating the expressed protein and,if required, renaturing the protein e.g., as described above.

Peptide Circularization.

In certain embodiments the peptides described herein (e.g., AMPs,compound AMPs, etc.) are circularized to produce cyclic peptides. Cyclicpeptides, as contemplated herein, include head/tail, head/side chain,tail/side chain, and side chain/side chain cyclized peptides. Inaddition, peptides contemplated herein include homodet, containing onlypeptide bonds, and heterodet containing in addition disulfide, ester,thioester-bonds, or other bonds.

The cyclic peptides can be prepared using virtually any art-knowntechnique for the preparation of cyclic peptides. For example, thepeptides can be prepared in linear or non-cyclized form usingconventional solution or solid phase peptide syntheses and cyclizedusing standard chemistries. Preferably, the chemistry used to cyclizethe peptide will be sufficiently mild so as to avoid substantiallydegrading the peptide. Suitable procedures for synthesizing the peptidesdescribed herein as well as suitable chemistries for cyclizing thepeptides are well known in the art.

In various embodiments cyclization can be achieved via direct couplingof the N- and C-terminus to form a peptide (or other) bond, but can alsooccur via the amino acid side chains. Furthermore it can be based on theuse of other functional groups, including but not limited to amino,hydroxy, sulfhydryl, halogen, sulfonyl, carboxy, and thiocarboxy. Thesegroups can be located at the amino acid side chains or be attached totheir N- or C-terminus.

Accordingly, it is to be understood that the chemical linkage used tocovalently cyclize the peptides of the invention need not be an amidelinkage. In many instances it may be desirable to modify the N- andC-termini of the linear or non-cyclized peptide so as to provide, forexample, reactive groups that may be cyclized under mild reactionconditions. Such linkages include, by way of example and not limitationamide, ester, thioester, CH₂—NH, etc. Techniques and reagents forsynthesizing peptides having modified termini and chemistries suitablefor cyclizing such modified peptides are well-known in the art.

Alternatively, in instances where the ends of the peptide areconformationally or otherwise constrained so as to make cyclizationdifficult, it may be desirable to attach linkers to the N- and/orC-termini to facilitate peptide cyclization. Of course, it will beappreciated that such linkers will bear reactive groups capable offorming covalent bonds with the termini of the peptide. Suitable linkersand chemistries are well-known in the art and include those previouslydescribed.

Cyclic peptides and depsipeptides (heterodetic peptides that includeester (depside) bonds as part of their backbone) have been wellcharacterized and show a wide spectrum of biological activity. Thereduction in conformational freedom brought about by cyclization oftenresults in higher receptor-binding affinities. Frequently in thesecyclic compounds, extra conformational restrictions are also built in,such as the use of D- and N-alkylated-amino acids, α,β-dehydro aminoacids or α,α-disubstituted amino acid residues.

Methods of forming disulfide linkages in peptides are well known tothose of skill in the art (see, e.g., Eichler and Houghten (1997)Protein Pept. Lett. 4: 157-164).

Reference may also be made to Marlowe (1993) Biorg. Med. Chem. Lett. 3:437-44 who describes peptide cyclization on TFA resin usingtrimethylsilyl (TMSE) ester as an orthogonal protecting group; Pallinand Tam (1995) J. Chem. Soc. Chem. Comm. 2021-2022) who describe thecyclization of unprotected peptides in aqueous solution by oximeformation; Algin et al. (1994) Tetrahedron Lett. 35: 9633-9636 whodisclose solid-phase synthesis of head-to-tail cyclic peptides vialysine side-chain anchoring; Kates et al. (1993) Tetrahedron Lett. 34:1549-1552 who describe the production of head-to-tail cyclic peptides bythree-dimensional solid phase strategy; Tumelty et al. (1994) J. Chem.Soc. Chem. Comm. 1067-1068, who describe the synthesis of cyclicpeptides from an immobilized activated intermediate, where activation ofthe immobilized peptide is carried out with N-protecting group intactand subsequent removal leading to cyclization; McMurray et al. (1994)Peptide Res. 7: 195-206) who disclose head-to-tail cyclization ofpeptides attached to insoluble supports by means of the side chains ofaspartic and glutamic acid; Hruby et al. (1994) Reactive Polymers 22:231-241) who teach an alternate method for cyclizing peptides via solidsupports; and Schmidt and Langer (1997) J. Peptide Res. 49: 67-73, whodisclose a method for synthesizing cyclotetrapeptides andcyclopentapeptides.

These methods of peptide cyclization are illustrative and non-limiting.Using the teaching provide herein, other cyclization methods will beavailable to one of skill in the art.

V. Identification/Verification of Active Peptides

The active AMPs and AMP constructs can be identified and/or validatedusing an in vitro screening assay. Indeed, in many instances the AMPsdescribed herein will be used in vitro as preservatives, topicalantimicrobial treatments, and the like. Additionally, despite certainapparent limitations of in vitro susceptibility tests, clinical dataindicate that a good correlation exists between minimal inhibitoryconcentration (MIC) test results and in vivo efficacy of antibioticcompounds (see, e.g., Murray et al. (1994) Antimicrobial SusceptibilityTesting, Poupard et al., eds., Plenum Press, New York; Knudsen et al.(1995) Antimicrob. Agents Chemother. 39(6): 1253-1258; and the like).Thus, AMPs useful for treating infections and diseases related theretoare also conveniently identified by demonstrated in vitro antimicrobialactivity against specified microbial targets, e.g., as illustrated inTable 3).

Typically, the in vitro antimicrobial activity of antimicrobial agentsis tested using standard NCCLS bacterial inhibition assays, or MIC tests(see, National Committee on Clinical Laboratory Standards “PerformanceStandards for Antimicrobial Susceptibility Testing,” NCCLS DocumentM100-S5 Vol. 14, No. 16, December 1994; “Methods for dilutionantimicrobial susceptibility test for bacteria that growaerobically-Third Edition,” Approved Standard M7-A3, National Committeefor Clinical Standards, Villanova, Pa.).

In certain embodiments the MIC assays are performed as described hereinin the Examples. For each organism, the media and growth conditionsutilized is detailed in Table 8. MIC tests were conducted in 96-wellplates with 100 μL of bacterial or yeast suspension added in each welland challenged with twofold serial dilutions of peptide starting at 50μM. After incubation 18-24 h, or in some cases 48 h, the lowestconcentration at which the peptide inhibited bacterial and/or fungalgrowth was noted as the MIC (observation of a clear well by visualinspection).

TABLE 8 List of organisms, growth conditions and starting inoculumconcentrations Starting Growth inoculum Organism Medium conditions(CFU/ml) A. baumannii LB 30° C., anaerobic 10⁵ A. naeslundii Columbiabroth 37° C., anaerobic 10⁵ A. niger Potato Dextrose 25° C., aerobic 10⁵Agar B. subtilis BHI 30° C., aerobic 10⁵ C. albicans MH 37° C., aerobic10⁵ C. difficile BHIS 37° C., anaerobic 10⁵ C. jeikeium TSB supplemented37° C., aerobic 10⁶ with 1% Tween-80 E. faecalis TSB supplemented 37°C., aerobic 10⁵ with 1% glucose E. coli LB 37° C., aerobic 10⁵ F.nucleatum Columbia broth 37° C., anaerobic 10⁵ M. furfur ATCC medium 37°C., aerobic 10⁵ 1072 P. gingivalis Columbia broth 37° C., anaerobic 10⁵P. aeruginosa MH 37° C., aerobic 10⁵ S. aureus MRSA BHI supplemented 37°C., aerobic 10⁵ with 2% glucose S. epidermidis BHI supplemented 37° C.,aerobic 10⁵ with 0.25% glucose S. mutans TH supplemented 37° C.,anaerobic 10⁵ with 1% sucrose S. pneumoniae TSB supplemented 37° C., 10%CO2 10⁶ with 5% yeast extract T. rubrum Potato Dextrose 25° C., aerobic10⁵ Agar

It will be appreciated that other assays as are well known in the art orthat will become apparent to those having skill in the art upon reviewof this disclosure may also be used to identify active AMPs. Such assaysinclude, for example, the assay described in Lehrer et al. (1988) J.Immunol. Meth., 108: 153 and Steinberg and Lehrer, “Designer Assays forAntimicrobial Peptides: Disputing the ‘One Size Fits All’ Theory,” In:Antibacterial Peptide Protocols, Shafer, Ed., Humana Press, N.J.Generally, active peptides of the invention will exhibit MICs (asmeasured using the assays described in the examples) of less than about100 μM, preferably less than about 80 or 60 μM, more preferably about 50μM or less, about 25 μM or less, or about 15 μM or less, or about 10 μMor less.

VI. Formulations.

Pharmaceutical Formulations.

In certain embodiments one or more active agents (e.g., antimicrobialpeptides (AMPs) or compound antimicrobial peptides described herein) areadministered to a mammal in need thereof, e.g., to a mammal sufferingfrom a microbial infection (e.g., bacterial or fungal infection) orprophylactically to prevent a microbial infection and/or to prevent orreduce the incidence or severity of dental caries.

The active agent(s) can be administered in the “native” form or, ifdesired, in the form of salts, esters, amides, prodrugs, derivatives,and the like, provided the salt, ester, amide, prodrug or derivative issuitable pharmacologically, i.e., effective in the present method(s).Salts, esters, amides, prodrugs and other derivatives of the activeagents can be prepared using standard procedures known to those skilledin the art of synthetic organic chemistry and described, for example, byMarch (1992) Advanced Organic Chemistry; Reactions, Mechanisms andStructure, 4th Ed. N.Y. Wiley-Interscience.

Methods of formulating such derivatives are known to those of skill inthe art. For example, the disulfide salts of a number of delivery agentsare described in PCT Publication WO 2000/059863. Similarly, acid saltsof therapeutic peptides, peptoids, or other mimetics, and can beprepared from the free base using conventional methodology thattypically involves reaction with a suitable acid. Generally, the baseform of the drug is dissolved in a polar organic solvent such asmethanol or ethanol and the acid is added thereto. The resulting salteither precipitates or can be brought out of solution by addition of aless polar solvent. Suitable acids for preparing acid addition saltsinclude, but are not limited to both organic acids, e.g., acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid,malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid,citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonicacid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, andthe like, as well as inorganic acids, e.g., hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike. An acid addition salt can be reconverted to the free base bytreatment with a suitable base. Certain particularly preferred acidaddition salts of the active agents herein include halide salts, such asmay be prepared using hydrochloric or hydrobromic acids. Conversely,preparation of basic salts of the active agents of this invention areprepared in a similar manner using a pharmaceutically acceptable basesuch as sodium hydroxide, potassium hydroxide, ammonium hydroxide,calcium hydroxide, trimethylamine, or the like. Particularly preferredbasic salts include alkali metal salts, e.g., the sodium salt, andcopper salts.

For the preparation of salt forms of basic drugs, the pKa of thecounterion is preferably at least about 2 pH lower than the pKa of thedrug. Similarly, for the preparation of salt forms of acidic drugs, thepKa of the counterion is preferably at least about 2 pH higher than thepKa of the drug. This permists the counterion to bring the solution's pHto a level lower than the pHmax to reach the salt plateau, at which thesolubility of salt prevails over the solubility of free acid or base.The generalized rule of difference in pKa units of the ionizable groupin the active pharmaceutical ingredient (API) and in the acid or base ismeant to make the proton transfer energetically favorable. When the pKaof the API and counterion are not significantly different, a solidcomplex may form but may rapidly disproportionate (i.e., break down intothe individual entities of drug and counterion) in an aqueousenvironment.

Preferably, the counterion is a pharmaceutically acceptable counterion.Suitable anionic salt forms include, but are not limited to acetate,benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate,edetate, edisylate, estolate, fumarate, gluceptate, gluconate,hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate,maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate,napsylate, nitrate, pamoate (embonate), phosphate and diphosphate,salicylate and disalicylate, stearate, succinate, sulfate, tartrate,tosylate, triethiodide, valerate, and the like, while suitable cationicsalt forms include, but are not limited to aluminum, benzathine,calcium, ethylene diamine, lysine, magnesium, meglumine, potassium,procaine, sodium, tromethamine, zinc, and the like.

Preparation of esters typically involves functionalization of hydroxyland/or carboxyl groups that are present within the molecular structureof the active agent. In certain embodiments, the esters are typicallyacyl-substituted derivatives of free alcohol groups, i.e., moieties thatare derived from carboxylic acids of the formula RCOOH where R is alky,and preferably is lower alkyl. Esters can be reconverted to the freeacids, if desired, by using conventional hydrogenolysis or hydrolysisprocedures.

Amides can also be prepared using techniques known to those skilled inthe art or described in the pertinent literature. For example, amidesmay be prepared from esters, using suitable amine reactants, or they maybe prepared from an anhydride or an acid chloride by reaction withammonia or a lower alkyl amine.

In various embodiments, the active agents identified herein are usefulfor parenteral, topical, oral, nasal (or otherwise inhaled), rectal, orlocal administration, such as by aerosol or transdermally, forprophylactic and/or therapeutic treatment of infection (e.g., microbialinfection) one or more of the pathologies/indications described herein(e.g., atherosclerosis and/or symptoms thereof).

The active agents of this invention can also be combined with apharmaceutically acceptable carrier (excipient) to form apharmacological composition. Pharmaceutically acceptable carriers cancontain one or more physiologically acceptable compound(s) that act, forexample, to stabilize the composition or to increase or decrease theabsorption of the active agent(s). Physiologically acceptable compoundscan include, for example, carbohydrates, such as glucose, sucrose, ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins, protection and uptake enhancerssuch as lipids, compositions that reduce the clearance or hydrolysis ofthe active agents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds, particularly of use in thepreparation of tablets, capsules, gel caps, and the like include, butare not limited to binders, diluent/fillers, disentegrants, lubricants,suspending agents, and the like.

In certain embodiments, to manufacture an oral dosage form (e.g., atablet), an excipient (e.g., lactose, sucrose, starch, mannitol, etc.),an optional disintegrator (e.g. calcium carbonate,carboxymethylcellulose calcium, sodium starch glycollate, crospovidoneetc.), a binder (e.g. alpha-starch, gum arabic, microcrystallinecellulose, carboxymethylcellulose, polyvinylpyrrolidone,hydroxypropylcellulose, cyclodextrin, etc.), and an optional lubricant(e.g., talc, magnesium stearate, polyethylene glycol 6000, etc.), forinstance, are added to the active component or components (e.g., activepeptide and salicylanilide) and the resulting composition is compressed.Where necessary the compressed product is coated, e.g., known methodsfor masking the taste or for enteric dissolution or sustained release.Suitable coating materials include, but are not limited toethyl-cellulose, hydroxymethylcellulose, polyoxyethylene glycol,cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, andEudragit (Rohm & Haas, Germany; methacrylic-acrylic copolymer).

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of pharmaceutically acceptable carrier(s),including a physiologically acceptable compound depends, for example, onthe route of administration of the active agent(s) and on the particularphysio-chemical characteristics of the active agent(s).

In certain embodiments the excipients are sterile and generally free ofundesirable matter. These compositions can be sterilized byconventional, well-known sterilization techniques. For various oraldosage form excipients such as tablets and capsules sterility is notrequired. The USP/NF standard is usually sufficient.

The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. Suitable unitdosage forms, include, but are not limited to powders, tablets, pills,capsules, lozenges, suppositories, patches, nasal sprays, injectibles,implantable sustained-release formulations, mucoadherent films, topicalvarnishes, lipid complexes, etc.

Pharmaceutical compositions comprising the peptides described herein canbe manufactured by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping or lyophilizing processes. Pharmaceutical compositions can beformulated in a conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries that facilitateprocessing of the active peptides into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

For topical administration the peptides of the invention may beformulated as solutions, gels, ointments, creams, suspensions, and thelike as are well-known in the art. Systemic formulations include, butare not limited to, those designed for administration by injection, e.g.subcutaneous, intravenous, intramuscular, intrathecal or intraperitonealinjection, as well as those designed for transdermal, transmucosal oralor pulmonary administration. For injection, the active agents describedherein can be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks solution, Ringer'ssolution, or physiological saline buffer and/or in certain emulsionformulations. The solution can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. In certain embodimentsthe active agent(s) can be provided in powder form for constitution witha suitable vehicle, e.g., sterile pyrogen-free water, before use. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated can be used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be readily formulated bycombining the active agent(s) with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. For oral solid formulations suchas, for example, powders, capsules and tablets, suitable excipientsinclude fillers such as sugars, such as lactose, sucrose, mannitol andsorbitol; cellulose preparations such as maize starch, wheat starch,rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP); granulating agents; and binding agents. Ifdesired, disintegrating agents may be added, such as the cross-linkedpolyvinylpyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate. If desired, solid dosage forms may be sugar-coated orenteric-coated using standard techniques.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like can be added. For buccaladministration, the compositions may take the form of tablets, lozenges,etc. formulated in conventional manner.

For administration by inhalation, the active agent(s) are convenientlydelivered in the form of an aerosol spray from pressurized packs or anebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

In various embodiments the active agent(s) can be formulated in rectalor vaginal compositions such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well known examples of delivery vehiclesthat may be used to deliver peptides of the invention. Certain organicsolvents such as dimethylsulfoxide also can be employed, althoughusually at the cost of greater toxicity. Additionally, the compounds maybe delivered using a sustained-release system, such as semipermeablematrices of solid polymers containing the therapeutic agent. Varioususes of sustained-release materials have been established and are wellknown by those skilled in the art. Sustained-release capsules may,depending on their chemical nature, release the compounds for a fewweeks up to over 100 days. Depending on the chemical nature and thebiological stability of the therapeutic reagent, additional strategiesfor protein stabilization may be employed.

In certain embodiments, the active agents of this invention areadministered to the oral cavity. This is readily accomplished by the useof lozenges, aersol sprays, mouthwash, coated swabs, and the like.

In certain embodiments, the active agent(s) of this invention areadministered topically, e.g., to the skin surface, to a topical lesionor wound, to a surgical site, and the like.

In certain embodiments the active agents of this invention areadministered systemically (e.g., orally, or as an injectable) inaccordance with standard methods well known to those of skill in theart. In other preferred embodiments, the agents, can also be deliveredthrough the skin using conventional transdermal drug delivery systems,i.e., transdermal “patches” wherein the active agent(s) are typicallycontained within a laminated structure that serves as a drug deliverydevice to be affixed to the skin. In such a structure, the drugcomposition is typically contained in a layer, or “reservoir,”underlying an upper backing layer. It will be appreciated that the term“reservoir” in this context refers to a quantity of “activeingredient(s)” that is ultimately available for delivery to the surfaceof the skin. Thus, for example, the “reservoir” may include the activeingredient(s) in an adhesive on a backing layer of the patch, or in anyof a variety of different matrix formulations known to those of skill inthe art. The patch may contain a single reservoir, or it may containmultiple reservoirs.

In one embodiment, the reservoir comprises a polymeric matrix of apharmaceutically acceptable contact adhesive material that serves toaffix the system to the skin during drug delivery. Examples of suitableskin contact adhesive materials include, but are not limited to,polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. Alternatively, the drug-containingreservoir and skin contact adhesive are present as separate and distinctlayers, with the adhesive underlying the reservoir which, in this case,may be either a polymeric matrix as described above, or it may be aliquid or hydrogel reservoir, or may take some other form. The backinglayer in these laminates, which serves as the upper surface of thedevice, preferably functions as a primary structural element of the“patch” and provides the device with much of its flexibility. Thematerial selected for the backing layer is preferably substantiallyimpermeable to the active agent(s) and any other materials that arepresent.

Other formulations for topical delivery include, but are not limited to,ointments, gels, sprays, fluids, and creams. Ointments are semisolidpreparations that are typically based on petrolatum or other petroleumderivatives. Creams containing the selected active agent are typicallyviscous liquid or semisolid emulsions, often either oil-in-water orwater-in-oil. Cream bases are typically water-washable, and contain anoil phase, an emulsifier and an aqueous phase. The oil phase, alsosometimes called the “internal” phase, is generally comprised ofpetrolatum and a fatty alcohol such as cetyl or stearyl alcohol; theaqueous phase usually, although not necessarily, exceeds the oil phasein volume, and generally contains a humectant. The emulsifier in a creamformulation is generally a nonionic, anionic, cationic or amphotericsurfactant. The specific ointment or cream base to be used, as will beappreciated by those skilled in the art, is one that will provide foroptimum drug delivery. As with other carriers or vehicles, an ointmentbase should be inert, stable, nonirritating and nonsensitizing.

As indicated above, various buccal, and sublingual formulations are alsocontemplated.

In certain embodiments, one or more active agents of the presentinvention can be provided as a “concentrate”, e.g., in a storagecontainer (e.g., in a premeasured volume) ready for dilution, or in asoluble capsule ready for addition to a volume of water, alcohol,hydrogen peroxide, or other diluent.

While the invention is described with respect to use in humans, it isalso suitable for animal, e.g., veterinary use. Thus certain preferredorganisms include, but are not limited to humans, non-human primates,canines, equines, felines, porcines, ungulates, largomorphs, and thelike.

The foregoing formulations and administration methods are intended to beillustrative and not limiting. It will be appreciated that, using theteaching provided herein, other suitable formulations and modes ofadministration can be readily devised.

Home Health Care Product Formulations.

In certain embodiments, one or more of the antimicrobial peptides (AMPs)and/or compound AMPs of the present invention are incorporated intohealthcare formulations, e.g., for home use. Such formulations include,but are not limited to toothpaste, mouthwash, tooth whitening strips orsolutions, contact lens storage, wetting, or cleaning solutions, dentalfloss, toothpicks, toothbrush bristles, oral sprays, oral lozenges,nasal sprays, aerosolizers for oral and/or nasal application, wounddressings (e.g., bandages), and the like.

The formulation of such health products is well known to those of skill,and the AMPs and/or compound AMPs of the present invention are simplyadded to such formulations in an effective dose (e.g., a prophylacticdose to inhibit dental carie formation, etc.).

For example, toothpaste formulations are well known to those of skill inthe art. Typically such formulations are mixtures of abrasives andsurfactants; anticaries agents, such as fluoride; tartar controlingredients, such as tetrasodium pyrophosphate and methyl vinylether/maleic anhydride copolymer; pH buffers; humectants, to preventdry-out and increase the pleasant mouth feel; and binders, to provideconsistency and shape (see, e.g., Table 9). Binders keep the solid phaseproperly suspended in the liquid phase to prevent separation of theliquid phase out of the toothpaste. They also provide body to thedentifrice, especially after extrusion from the tube onto thetoothbrush.

TABLE 9 Typical components of toothpaste. Ingredients Wt % Humectants40-70 Water  0-50 Buffers/salts/tartar 0.5-10  control Organicthickeners 0.4-2   (gums) Inorganic thickeners  0-12 Abrasives 10-50Actives (e.g., triclosan) 0.2-1.5 Surfactants 0.5-2   Flavor andsweetener 0.8-1.5 Fluoride sources provide 1000-15000 ppm fluorine.

Table 10 lists typical ingredients used in formulations; the finalcombination will depend on factors such as ingredient compatibility andcost, local customs, and desired benefits and quality to be delivered inthe product. It will be recognized that one or more AMPs and/or compoundAMPs of the present invention can simply be added to such formulationsor used in place of one or more of the other ingredients.

TABLE 10 List of typical ingredients. Tartar Inorganic Control GumsThickeners Abrasives Surfactants Humectants Ingredient Sodium SilicaHydrated Sodium Glycerine Tetrasodium carboxymethyl thickeners silicalauryl sulfate pyrophosphate cellulose Cellulose ethers Sodium DicalciumSodium N- Sorbitol Gantrez S-70 aluminum phosphate lauryl silicatesdigydrate sarcosinate Xanthan Gum Clays Calcium Pluronics PropyleneSodium tri- carbonate glycol polyphosphate Carrageenans Sodium Xylitolbicarbonate Sodium alginate Calcium Sodium Polyethylene pyrophosphatelauryl glycol sulfoacetate Carbopols Alumina

One illustrative formulation described in U.S. Pat. No. 6,113,887comprises (1) a water-soluble bactericide selected from the groupconsisting of pyridinium compounds, quaternary ammonium compounds andbiguanide compounds in an amount of 0.001% to 5.0% by weight, based onthe total weight of the composition; (2) a cationically-modifiedhydroxyethylcellulose having an average molecular weight of 1,000,000 orhigher in the hydroxyethylcellulose portion thereof and having acationization degree of 0.05 to 0.5 mol/glucose in an amount of 0.5% to5.0% by weight, based on the total weight of the composition; (3) asurfactant selected from the group consisting of polyoxyethylenepolyoxypropylene block copolymers and alkylolamide compounds in anamount of 0.5% to 13% by weight, based on the total weight of thecomposition; and (4) a polishing agent of the non-silica type in anamount of 5% to 50% by weight, based on the total weight of thecomposition. In certain embodiments, the AMPs and/or compound AMPs ofthis invention can be used in place of the bactericide or in combinationwith the bactericide.

Similarly, mouthwash formulations are also well known to those of skillin the art. Thus, for example, mouthwashes containing sodium fluorideare disclosed in U.S. Pat. Nos: 2,913,373, 3,975,514, and 4,548,809, andin US Patent Publications US 2003/0124068 A1, US 2007/0154410 A1, andthe like. Mouthwashes containing various alkali metal compounds are alsoknown: sodium benzoate (WO 9409752); alkali metal hypohalite (US20020114851A1); chlorine dioxide (CN 1222345); alkali metal phosphate(US 2001/0002252 A1, US 2003/0007937 A1); hydrogen sulfate/carbonate (JP8113519); cetylpyridium chloride (CPC) (see, e.g., U.S. Pat. No.6,117,417, U.S. Pat. No. 5,948,390, and JP 2004051511). Mouthwashescontaining higher alcohol (see, e.g., US 2002/0064505 A1, US2003/0175216 A1); hydrogen peroxide (see, e.g., CN 1385145); CO₂ gasbubbles (see, e.g., JP 1275521 and JP 2157215) are also known. Incertain embodiments, these and other mouthwash formulations can furthercomprise one or more of the AMPs or compound AMPs of this invention.

Contact lens storage, wetting, or cleaning solutions, deodorants, dentalfloss, toothpicks, toothbrush bristles, oral sprays, oral lozenges,nasal sprays, and aerosolizers for oral and/or nasal application, andthe like are also well known to those of skill in the art and canreadily be adapted to incorporate one or more AMPs and/or compound AMPsof the present invention.

The foregoing home healthcare formulations and/or devices are meant tobe illustrative and not limiting. Using teaching provided herein, theAMPs and/or compound AMPs of the present invention can readily beincorporated into other products.

Effective Dosages

The AMPs compound AMPs and other constructs described herein willgenerally be used in an amount effective to achieve the intendedpurpose. Of course, it is to be understood that the amount used willdepend on the particular application. For example, for use as adisinfectant or preservative, an antimicrobially effective amount of anantimicrobial peptide, or composition thereof, is applied or added tothe material to be disinfected or preserved. By antimicrobiallyeffective amount is meant an amount of peptide or composition thatinhibits the growth and/or proliferation of, or is lethal to, a targetmicrobe population. While the actual antimicrobially effective amountwill depend on a particular application, for use as a disinfectant orpreservative the peptides, or compositions thereof, are usually added orapplied to the material to be disinfected or preserved in relatively lowamounts. Typically, the peptide comprises less than about 20%, 15%, 10%,or 5% by weight of the disinfectant solution or material to bepreserved, preferably less than about 1% by weight and more preferablyless than about 0.1% by weight. An ordinarily skilled artisan will beable to determine antimicrobially effective amounts of particularpeptides for particular applications without undue experimentationusing, for example, the in vitro assays provided in the examples.

In certain therapeutic applications, the compositions of this inventionare administered, e.g., topically administered or administered to theoral or nasal cavity, to a patient suffering from infection or at riskfor infection or prophylactically to prevent infection. In certainembodiments the administration is to prevent dental caries and/orperiodontal disease, and/or other pathologies of the teeth or oralmucosa characterized by microbial infection.

More generally, the composition (e.g., AMP, compound AMP, etc.) isadministered therapeutically to kill and/or to inhibit the growth and/orproliferation of a microorganism and/or a biofilm comprising one or moremicroorganisms. Similarly the composition can be administeredprophylactically to reduce the infectivity of a microorganism and/or toprevent/inhibit the growth and/or proliferation of a microorganismand/or a biofilm comprising the microorganism.

An amount adequate to kill and/or inhibit the growth and/orproliferation of a microorganism or sufficient to prevent and/or cureand/or at least partially prevent or arrest a disease and/or itscomplications is defined as a “therapeutically effective dose.” Amountseffective for this use will depend upon the severity of the disease andthe general state of the patient's health. Single or multipleadministrations of the compositions may be administered depending on thedosage and frequency as required and tolerated by the patient. In anyevent, the composition should provide a sufficient quantity of theactive agents of the formulations of this invention to effectively treat(ameliorate one or more symptoms in) the patient.

The concentration of active agent(s) can vary widely, and will beselected primarily based on activity of the active ingredient(s), bodyweight and the like in accordance with the particular mode ofadministration selected and the patient's needs. Concentrations,however, will typically be selected to provide dosages ranging fromabout 0.1 or 1 mg/kg/day to about 50 mg/kg/day and sometimes higher.Typical dosages range from about 3 mg/kg/day to about 3.5 mg/kg/day,preferably from about 3.5 mg/kg/day to about 7.2 mg/kg/day, morepreferably from about 7.2 mg/kg/day to about 11.0 mg/kg/day, and mostpreferably from about 11.0 mg/kg/day to about 15.0 mg/kg/day. In certainpreferred embodiments, dosages range from about 10 mg/kg/day to about 50mg/kg/day. In certain embodiments, dosages range from about 20 mg toabout 50 mg given orally twice daily. It will be appreciated that suchdosages may be varied to optimize a therapeutic and/or phophylacticregimen in a particular subject or group of subjects. Determination of atherapeutically effective amount is well within the capabilities ofthose skilled in the art, especially in light of the detailed disclosureprovided herein.

As in the case of disinfectants and preservatives, for topicaladministration to treat or prevent bacterial, yeast, fungal or otherinfections a therapeutically effective dose can be determined using, forexample, the in vitro assays provided in the examples. The treatment maybe applied while the infection is visible, or even when it is notvisible. An ordinarily skilled artisan will be able to determinetherapeutically effective amounts to treat topical infections withoutundue experimentation.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating cyclic peptideconcentration range that includes the I₅₀ as determined in cell culture(i.e., the concentration of test compound that is lethal to 50% of acell culture), the MIC, as determined in cell culture (i.e., the minimalinhibitory concentration for growth) or the I₁₀₀ as determined in cellculture (i.e., the concentration of peptide that is lethal to 100% of acell culture). Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data. In certain embodiments dosage amount andinterval can be adjusted individually to provide plasma levels of theactive peptide which are sufficient to maintain therapeutic effect.

In cases of local administration or selective uptake, the effectivelocal concentration of peptide may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

The antimicrobial therapy may be repeated intermittently whileinfections are detectable or even when they are not detectable. Thetherapy may be provided alone or in combination with other drugs, suchas for example antibiotics or other antimicrobial peptides.

Toxicity

Preferably, a therapeutically effective dose of the AMPs and otherconstructs described herein will provide therapeutic benefit withoutcausing substantial toxicity.

Toxicity can be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., by determining the LD₅₀ (thedose lethal to 50% of the population) or the LD₁₀₀ (the dose lethal to100% of the population). The dose ratio between toxic and therapeuticeffect is the therapeutic index. Compounds that exhibit high therapeuticindices are preferred, particularly for in vivo applications. The dataobtained from cell culture assays and animal studies can be used informulating a dosage range that is not toxic for use in human. Thedosage of the peptides described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition (see,e.g., Fingl et al. (1975) In: The Pharmacological Basis of Therapeutics,Ch. 1, p. 1).

VII. Kits.

In another embodiment this invention provides kits for the inhibition ofan infection and/or for the treatment and/or prevention of dental cariesin a mammal and/or the inhibition of biofilms (e.g., on a prosthetic ormedical implant). The kits typically comprise a container containing oneor more of the active agents (i.e., antimicrobial peptides or compoundantimicrobial peptides) described herein. In certain embodiments theactive agent(s) can be provided in a unit dosage formulation (e.g.,suppository, tablet, caplet, patch, etc.) and/or may be optionallycombined with one or more pharmaceutically acceptable excipients.

In certain embodiments the kits comprise one or more of the homehealthcare product formulations described herein (e.g., toothpaste,mouthwash, tooth whitening strips or solutions, contact lens storage,wetting, or cleaning solutions, dental floss, toothpicks, toothbrushbristles, oral sprays, oral lozenges, nasal sprays, aerosolizers fororal and/or nasal application, and the like).

In certain embodiments the kits comprise one or more of the disinfectantformulations described herein.

In addition, the kits optionally include labeling and/or instructionalmaterials providing directions (i.e., protocols) for the practice of themethods or use of the “therapeutics” or “prophylactics” of thisinvention. Preferred instructional materials describe the use of one ormore active agent(s) of this invention to therapeutically orprophylactically to inhibit or prevent infection and/or to inhibit theformation of dental caries. The instructional materials may also,optionally, teach preferred dosages/therapeutic regiment, counterindications and the like.

While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Synthesis and Screening of Antimicrobial Peptides

Peptides were synthesized utilizing standard solid-phase synthesismethods (Fmoc or Boc chemistries). In certain embodiments, peptides weresynthesized using Fmoc (9-fluorenylmethoxy carbonyl) solid-phasesynthesis utilizing single or double coupling cycles at 0.01 to 0.25mmol scales. Briefly, N-terminal deprotection was conducted in 20% (v/v)piperidine/N-methylpyrrolidone (NMP) for 3 min followed by eight washeswith NMP. For the coupling cycle, amino acids were solubilized in 0.45 MN-hydroxybenzotrazole (HOBt)/HBTU(O-benzotriazole-N,N,N,N-tetramethyl-uronium hexafluoro-phosphate) indimethylformamide (DMF) with 0.9 M diisopropyl ethylamine (DIEA)(1:1:1:2 ratio Fmoc amino acid:HOB:HBTU:DIEA) before being added to theresin for 30 min, followed by 10 NMP rinses. As needed, peptides werethen labeled N-terminally with 4-molar excess fluorescent dyes orblocking groups in HOBt/HBTU/DIEA coupling solution with shaking atambient temperature for 24 h followed by 10 rinses in dichloromethane(DCM).

After synthesis, the resin was washed 4× in DCM, 1× in MeOH, and dried24 h under desiccant vacuum. Completed peptides were cleaved from theresin with 90% trifluoroacetic acid (TFA) and appropriate scavengingreagents. In some examples, D or L form amino acids, or a mixture ofboth, were used in synthesis. All peptides were purified byreverse-phase high-performance liquid chromatography (HPLC). Peptidemass was determined by electrospray ionization (ESI) mass spectrometry.Peptides were utilized in TFA salt form, acetate salt form, or HCl saltforms. Peptides were then screened for antimicrobial activity utilizingan MIC assay.

In certain embodiments the MIC assays are performed as follows: For eachorganism, the media and growth conditions utilized is detailed in Table8. MIC tests were conducted in 96-well plates with 100 μL of bacterialor yeast suspension added in each well and challenged with twofoldserial dilutions of peptide starting at 50 μM. After incubation 18-24 h,or in some cases 48 h, the lowest concentration at which the peptideinhibited bacterial and/or fungal growth was noted as the MIC(observation of a clear well by visual inspection).

In some cases killing kinetic experiments were then conducted. Killingkinetics of PF-S028 L and D versions against M furfur were determined asdescribed in Eckert et al. (2006) Antimicrob Agents Chemother., 50:1480-1488. Briefly, a log phase culture of M. furfur ATCC 14521 wasdiluted to 10⁶ cells/ml in ATCC medium 1072. To the reaction tubes 5 μMof either PF-S028 L or PF-S028 D were added. A reaction tube to which 4μl of 50% methanol was added served as the negative control. Reactiontubes were stored at 30° C. and at indicated intervals a sample wasremoved from the reaction tube and placed in a recovery tube wherepeptide was removed by dilution. An aliquot from the recovery tube wasplated on ATCC medium 1072 agar plates and incubated at 30° C. untilvisible colonies formed. Colony forming units were calculated for thenegative control (no peptide), PF-S028 L, and PF-S028 D treatedcultures. After 15 min of treatment, PF-S028 D killed over 99% of the M.furfur. PF-S028 L killed 39% (FIG. 1).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. An isolated antimicrobial peptide having antimicrobial activityagainst at least one kind of bacteria, fungus, or yeast, saidantimicrobial peptide ranging in length up to 60 amino acids andcomprising one or more amino acid sequences independently selected fromgroup consisting of GSVIKKRRKRMSKKKHRKMLRRTRVQRRKLGK (PF-S028, SEQ IDNO:1), NYRLVNAIFSKIFKKKFIKF (PF-C252, SEQ ID NO:2),YIQFHLNQQPRPKVKKIKIFL (PF-531, SEQ ID NO:3),GSVIKKRRKRMAKKKHRKLLKKTRIQRRRAGK (PF-527, SEQ ID NO:4),MRFGSLALVAYDSAIKHSWPRPSSVRRLRM (PF-672, SEQ ID NO:5),FESKILNASKELDKEKKVNTALSFNSHQDFAKAYQNGKI (PF-606, SEQ ID NO:6),KGKSLMPLLKQINQWGKLYL (PF-C239, SEQ ID NO:7), WSRVPGHSDTGWKVWHRW (PF-547,SEQ ID NO:8), MGIIAGIIKFIKGLIEKFTGK (PF-006, SEQ ID NO:9),ILNKKPKLPLWKLGKNYFRRFYVLPTFLA (PF-C287, SEQ ID NO:10), RESKLIAMADMIRRRI(PF-545, SEQ ID NO:11), LDPLEPRIAPPGDRSHQGAPACHRDPLRGRSARDAER (PF-0019,SEQ ID NO:12), MPVSKKRYMLSSAYATALGICYGQVATDEKESEITAIPDLLDYLSVEEYLL(PF-C163, SEQ ID NO:13), LSLATFAKIFMTRSNWSLKRFNRL (PF-278, SEQ IDNO:14), MIRIRSPTKKKLNRNSISDWKSNTSGRFFY (PF-283, SEQ ID NO:15),MKRRRCNWCGKLFYLEEKSKEAYCCKECRKKAKKVKK (PF-307, SEQ ID NO:16),VLPFPAIPLSRRRACVAAPRPRSRQRAS (PF-168, SEQ ID NO:17),KNKKQTDILEKVKEILDKKKKTKSVGQKLY (PF-538, SEQ ID NO:18), SLQSQLGPCLHDQRH(PF-448, SEQ ID NO:19), WKRLWPARILAGHSRRRMRWMVVWRYFAAT (PF-C021, SEQ IDNO:20), KFQGEFTNIGQSYIVSASHMSTSLNTGK (PF-583, SEQ ID NO:21),TKKIELKRFVDAFVKKSYENYILERELKKLIKAINEELPTK (PF-600, SEQ ID NO:22),KFSDQIDKGQDALKDKLGDL (PF-525, SEQ ID NO:23), LSEMERRRLRKRA (PF-529, SEQID NO:24), RRGCTERLRRMARRNAWDLYAEHFY (PF-148, SEQ ID NO:25),SKFKVLRKIIIKEYKGELMLSIQKQR (PF-530, SEQ ID NO:26), FELVDWLETNLGKILKSKSA(PF-522, SEQ ID NO:27), LVLRICTDLFTFIKWTIKQRKS (PF-497, SEQ ID NO:28),VYSFLYVLVIVRKLLSMKKRIERL (PF-499, SEQ ID NO:29), GIVLIGLKLIPLLANVLR(PF-322, SEQ ID NO:30), VMQSLYVKPPLILVTKLAQQN (PF-511, SEQ ID NO:31),SFMPEIQKNTIPTQMK (PF-512, SEQ ID NO:32),LGLTAGVAYAAQPTNQPTNQPTNQPTNQPTNQPTNQPRW (PF-520, SEQ ID NO:33),CGKLLEQKNFFLKTR (PF-521, SEQ ID NO:34), ASKQASKQASKQASKQASKQASRSLKNHLL(PF-523, SEQ ID NO:35), PDAPRTCYHKPILAALSRIVVTDR (PF-524, SEQ ID NO:36),NYAVVSHT (PF-209, SEQ ID NO:37), ILVLLALQVELDSKFQY (PF-C157, SEQ IDNO:38), YVNYNQSFNSGW (PF-C220, SEQ ID NO:39), andFQKPFTGEEVEDFQDDDEIPTII (PF-437, SEQ ID NO:40).
 2. The antimicrobialpeptide of claim 1, wherein the amino acid sequence of said peptideconsists of a sequence selected from group consisting ofGSVIKKRRKRMSKKKHRKMLRRTRVQRRKLGK (PF-5028, SEQ ID NO:1),NYRLVNAIFSKIFKKKFIKF (PF-C252, SEQ ID NO:2), YIQFHLNQQPRPKVKKIKIFL(PF-531, SEQ ID NO:3), GSVIKKRRKRMAKKKHRKLLKKTRIQRRRAGK (PF-527, SEQ IDNO:4), MRFGSLALVAYDSAIKHSWPRPSSVRRLRM (PF-672, SEQ ID NO:5),FESKILNASKELDKEKKVNTALSFNSHQDFAKAYQNGKI (PF-606, SEQ ID NO:6),KGKSLMPLLKQINQWGKLYL (PF-C239, SEQ ID NO:7), WSRVPGHSDTGWKVWHRW (PF-547,SEQ ID NO:8), MGIIAGIIKFIKGLIEKFTGK (PF-006, SEQ ID NO:9),ILNKKPKLPLWKLGKNYFRRFYVLPTFLA (PF-C287, SEQ ID NO:10), RESKLIAMADMIRRRI(PF-545, SEQ ID NO:11), LDPLEPRIAPPGDRSHQGAPACHRDPLRGRSARDAER (PF-0019,SEQ ID NO:12), MPVSKKRYMLSSAYATALGICYGQVATDEKESEITAIPDLLDYLSVEEYLL(PF-C163, SEQ ID NO:13), LSLATFAKIFMTRSNWSLKRFNRL (PF-278, SEQ IDNO:14), MIRIRSPTKKKLNRNSISDWKSNTSGRFFY (PF-283, SEQ ID NO:15),MKRRRCNWCGKLFYLEEKSKEAYCCKECRKKAKKVKK (PF-307, SEQ ID NO:16),VLPFPAIPLSRRRACVAAPRPRSRQRAS (PF-168, SEQ ID NO:17),KNKKQTDILEKVKEILDKKKKTKSVGQKLY (PF-538, SEQ ID NO:18), SLQSQLGPCLHDQRH(PF-448, SEQ ID NO:19), WKRLWPARILAGHSRRRMRWMVVWRYFAAT (PF-C021, SEQ IDNO:20), KFQGEFTNIGQSYIVSASHMSTSLNTGK (PF-583, SEQ ID NO:21),TKKIELKRFVDAFVKKSYENYILERELKKLIKAINEELPTK (PF-600, SEQ ID NO:22),KFSDQIDKGQDALKDKLGDL (PF-525, SEQ ID NO:23), LSEMERRRLRKRA (PF-529, SEQID NO:24), RRGCTERLRRMARRNAWDLYAEHFY (PF-148, SEQ ID NO:25),SKFKVLRKIIIKEYKGELMLSIQKQR (PF-530, SEQ ID NO:26), FELVDWLETNLGKILKSKSA(PF-522, SEQ ID NO:27), LVLRICTDLFTFIKWTIKQRKS (PF-497, SEQ ID NO:28),VYSFLYVLVIVRKLLSMKKRIERL (PF-499, SEQ ID NO:29), GIVLIGLKLIPLLANVLR(PF-322, SEQ ID NO:30), VMQSLYVKPPLILVTKLAQQN (PF-511, SEQ ID NO:31),SFMPEIQKNTIPTQMK (PF-512, SEQ ID NO:32),LGLTAGVAYAAQPTNQPTNQPTNQPTNQPTNQPTNQPRW (PF-520, SEQ ID NO:33),CGKLLEQKNFFLKTR (PF-521, SEQ ID NO:34), ASKQASKQASKQASKQASKQASRSLKNHLL(PF-523, SEQ ID NO:35), PDAPRTCYHKPILAALSRIVVTDR (PF-524, SEQ ID NO:36),NYAVVSHT (PF-209, SEQ ID NO:37), ILVLLALQVELDSKFQY (PF-C157, SEQ IDNO:38), YVNYNQSFNSGW (PF-C220, SEQ ID NO:39), andFQKPFTGEEVEDFQDDDEIPTII (PF-437, SEQ ID NO:40).
 3. The antimicrobialpeptide of claim 1, wherein said peptide is effective to kill or inhibitthe growth or proliferation of a yeast and/or fungus, wherein the aminoacid sequence of said peptide comprises one or more sequences selectedfrom the group consisting of PF-148, PF-168, PF-448, PF-525, PF-527,PF-529, PF-531, PF-545, PF-672, PF-0019, PF-278, PF-307, PF-672,PF-C021, PF-C157, PF-C220, PF-C252, PF-C287, PF-5028, PF-168, PF-278,PF-283, PF-307, PF-527, PF-531, PF-547, PF-672, PF-0019, PF-C021,PF-C252, and PF-5028.
 4. The antimicrobial peptide according to claim 3,wherein said yeast and/or fungus is selected from the group consistingof A. niger, C. albicans, T. rubrum, and M. furfur.
 5. The antimicrobialpeptide of claim 1, wherein said peptide is effective to kill or inhibitthe growth or proliferation of a bacterium, wherein the amino acidsequence of said peptide comprises one or more sequences selected fromthe group consisting of PF-006, PF-530, PF-531, PF-538, PF-C163,PF-C239, PF-C252, PF-C287, PF-006, PF-148, PF-283, PF-307, PF-322,PF-497, PF-499, PF-527, PF-531, PF-545, PF-547, PF-672, PF-5028, PF-522,PF-531, PF-538, PF-600, PF-606, PF-672, PF-C239, PF-C252, PF-006,PF-168, PF-209, PF-527, PF-545, PF-583, PF-606, PF-672, PF-C252,PF-5028, PF-5028, PF-C252, PF-531, PF-527, PF-5028, PF-C163, PF-C239,PF-C252, PF-C287, PF-278, PF-283, PF-527, PF-531, PF-583, PF-606,PF-672, PF-C163, PF-C252, PF-5028, PF-531, PF-547, PF-601, PF-0019,PF-C239, PF-C252, PF-S028, PF-437, PF-448, PF-511, PF-512, PF-520,PF-521, PF-523, PF-524, PF-525, PF-529, PF-600, and PF-606.
 6. Theantimicrobial peptide of claim 5, wherein said bacterium is selectedfrom the group consisting of A. naeslundii, S. mutans, B. subtilis,MRSA, C. difficile, S. epidermidis, S. pneumoniae, E. faecalis, P.gingivalis, E. coli, P. aeruginosa, A. baumannii, and F. nucleatum. 7.The antimicrobial peptide of claim 5, wherein said peptide is effectiveto kill or inhibit the growth or proliferation of a gram positivebacterium, wherein the amino acid sequence of said peptide comprises oneor more sequences selected from the group consisting of PF-006, PF-148,PF-168, PF-209, PF-278, PF-283, PF-307, PF-322, PF-437, PF-448, PF-497,PF-499, PF-511, PF-512, PF-520, PF-521, PF-522, PF-523, PF-524, PF-525,PF-527, PF-529, PF-531, PF-538, PF-545, PF-547, PF-583, PF-600, PF-601,PF-606, PF-672, PF-0019, PF-C163, PF-C239, PF-C252, PF-C287, PF-S028. 8.The antimicrobial peptide of claim 7, wherein said gram positivebacterium is selected from the group consisting of A. naeslundii, S.mutans, B. subtilis, MRSA, C. difficile, S. epidermidis, S. pneumoniae,and E. faecalis.
 9. The antimicrobial peptide of claim 5, wherein saidpeptide is effective to kill or inhibit the growth or proliferation ofgram negative bacterium wherein the amino acid sequence of said peptidecomprises one or more sequences selected from the group consisting ofPF-006, PF-527, PF-530, PF-531, PF-538, PF-600, PF-606, PF-C163,PF-C239, PF-C252, PF-C287, and PF-S028.
 10. The antimicrobial peptide ofclaim 9, wherein said negative bacterium is selected from the groupconsisting of P. gingivalis, E. coli, P. aeruginosa, A. baumannii, andF. nucleatum.
 11. The antimicrobial peptide according to of claim 1,wherein said peptide comprises all “L” amino acids.
 12. Theantimicrobial peptide of claim 1, wherein said peptide comprises all “D”amino acids.
 13. The antimicrobial peptide of claim 1, wherein saidpeptide is a β peptide.
 14. The antimicrobial peptide of claim 1,wherein said peptide comprises one or more protecting groups. 15-17.(canceled)
 18. The antimicrobial peptide of claim 1, wherein saidpeptide is in a pharmaceutically acceptable carrier.
 19. Theantimicrobial peptide according to claim 18, wherein said carrier issuitable for administration via a route selected from the groupconsisting of topical administration, aerosol administration,administration via inhalation, oral administration, systemic IVapplication, ocular administration, and rectal administration.
 20. Achimeric construct comprising an antimicrobial peptide of claim 1attached to a targeting peptide comprising one or more of the amino acidsequences in Table
 6. 21. The construct of claim 20, wherein saidtargeting peptide is chemically conjugated to said antimicrobialpeptide.
 22. The construct of claim 21, wherein said targeting peptideis chemically conjugated to said antimicrobial peptide through anon-peptide linker shown in Table
 4. 23. The construct of claim 22,wherein said targeting peptide is coupled to said antimicrobial peptidevia a peptide linker.
 24. The construct of claim 21, wherein saidtargeting peptide is coupled to said antimicrobial peptide via a peptidelinker shown in Table
 4. 25. The construct of claim 20, wherein saidtargeting peptide and/or said antimicrobial peptide comprises all “L”amino acids.
 26. The construct claim 20, wherein said targeting peptideand/or said antimicrobial peptide comprises all “D” amino acids.
 27. Theconstruct claim 20, wherein said targeting peptide and/or saidantimicrobial peptide comprises a mixture of “L” and “D” amino acids.28. The construct claim 20, wherein said targeting peptide and/or saidantimicrobial peptide is a 0 peptide.
 29. The claim 20, wherein saidtargeting peptide and/or said antimicrobial peptide bears one or moreprotecting groups. 30-31. (canceled)
 32. An antimicrobial compositioneffective to kill and/or to inhibit the growth and/or proliferation of amicroorganism and/or to inhibit the formation and/or growth and/ormaintenance of a biofilm comprising said microorganism, said compositioncomprising one or more peptides, the amino acid sequences of saidpeptides comprising one or more sequences selected from the groupconsisting of GSVIKKRRKRMSKKKHRKMLRRTRVQRRKLGK (PF-S028, SEQ ID NO:1),NYRLVNAIFSKIFKKKFIKF (PF-C252, SEQ ID NO:2), YIQFHLNQQPRPKVKKIKIFL(PF-531, SEQ ID NO:3), GSVIKKRRKRMAKKKHRKLLKKTRIQRRRAGK (PF-527, SEQ IDNO:4), MRFGSLALVAYDSAIKHSWPRPSSVRRLRM (PF-672, SEQ ID NO:5),FESKILNASKELDKEKKVNTALSFNSHQDFAKAYQNGKI (PF-606, SEQ ID NO:6),KGKSLMPLLKQINQWGKLYL (PF-C239, SEQ ID NO:7), WSRVPGHSDTGWKVWHRW (PF-547,SEQ ID NO:8), MGIIAGIIKFIKGLIEKFTGK (PF-006, SEQ ID NO:9),ILNKKPKLPLWKLGKNYFRRFYVLPTFLA (PF-C287, SEQ ID NO:10), RESKLIAMADMIRRRI(PF-545, SEQ ID NO:11), LDPLEPRIAPPGDRSHQGAPACHRDPLRGRSARDAER (PF-0019,SEQ ID NO:12), MPVSKKRYMLSSAYATALGICYGQVATDEKESEITAIPDLLDYLSVEEYLL(PF-C163, SEQ ID NO:13), LSLATFAKIFMTRSNWSLKRFNRL (PF-278, SEQ IDNO:14), MIRIRSPTKKKLNRNSISDWKSNTSGRFFY (PF-283, SEQ ID NO:15),MKRRRCNWCGKLFYLEEKSKEAYCCKECRKKAKKVKK (PF-307, SEQ ID NO:16),VLPFPAIPLSRRRACVAAPRPRSRQRAS (PF-168, SEQ ID NO:17),KNKKQTDILEKVKEILDKKKKTKSVGQKLY (PF-538, SEQ ID NO:18), SLQSQLGPCLHDQRH(PF-448, SEQ ID NO:19), WKRLWPARILAGHSRRRMRWMVVWRYFAAT (PF-C021, SEQ IDNO:20), KFQGEFTNIGQSYIVSASHMSTSLNTGK (PF-583, SEQ ID NO:21),TKKIELKRFVDAFVKKSYENYILERELKKLIKAINEELPTK (PF-600, SEQ ID NO:22),KFSDQIDKGQDALKDKLGDL (PF-525, SEQ ID NO:23), LSEMERRRLRKRA (PF-529, SEQID NO:24), RRGCTERLRRMARRNAWDLYAEHFY (PF-148, SEQ ID NO:25),SKFKVLRKIIIKEYKGELMLSIQKQR (PF-530, SEQ ID NO:26), FELVDWLETNLGKILKSKSA(PF-522, SEQ ID NO:27), LVLRICTDLFTFIKWTIKQRKS (PF-497, SEQ ID NO:28),VYSFLYVLVIVRKLLSMKKRIERL (PF-499, SEQ ID NO:29), GIVLIGLKLIPLLANVLR(PF-322, SEQ ID NO:30), VMQSLYVKPPLILVTKLAQQN (PF-511, SEQ ID NO:31),SFMPEIQKNTIPTQMK (PF-512, SEQ ID NO:32),LGLTAGVAYAAQPTNQPTNQPTNQPTNQPTNQPTNQPRW (PF-520, SEQ ID NO:33),CGKLLEQKNFFLKTR (PF-521, SEQ ID NO:34), ASKQASKQASKQASKQASKQASRSLKNHLL(PF-523, SEQ ID NO:35), PDAPRTCYHKPILAALSRIVVTDR (PF-524, SEQ ID NO:36),NYAVVSHT (PF-209, SEQ ID NO:37), ILVLLALQVELDSKFQY (PF-C157, SEQ IDNO:38), YVNYNQSFNSGW (PF-C220, SEQ ID NO:39), andFQKPFTGEEVEDFQDDDEIPTII (PF-437, SEQ ID NO:40).
 33. The compositionaccording to claim 19, wherein said composition is effective to kill orinhibit the growth and/or proliferation of a yeast or fungus, and saidcomposition comprises one or more peptides, the amino acid sequences ofsaid peptides comprising one or more sequences selected from the groupconsisting of PF-148, PF-168, PF-448, PF-525, PF-527, PF-529, PF-531,PF-545, PF-672, PF-0019, PF-278, PF-307, PF-672, PF-C021, PF-C157,PF-C220, PF-C252, PF-C287, PF-5028, PF-168, PF-278, PF-283, PF-307,PF-527, PF-531, PF-547, PF-672, PF-0019, PF-C021, PF-C252, and PF-5028.34. The composition according to claim 33, wherein said composition iseffective to kill or inhibit the growth and/or proliferation ofAspergillus niger and said composition comprises one or more peptides,the amino acid sequences of said peptides comprising one or moresequences selected from the group consisting of PF-148, PF-168, PF-448,PF-525, PF-527, PF-529, PF-531, PF-545, PF-672, and PF-0019.
 35. Thecomposition according to claim 33, wherein said composition is effectiveto kill or inhibit the growth and/or proliferation of Candida albicansand said composition comprises one or more peptides, the amino acidsequences of said peptides comprising one or more sequences selectedfrom the group consisting of PF-278, PF-307, PF-672, PF-C021, PF-C157,PF-C220, PF-C252, and PF-C287.
 36. The composition according to claim33, wherein said composition is effective to kill or inhibit the growthand/or proliferation of Malassezia furfur and said composition comprisesa peptide comprising the amino acid sequence of PF-S028.
 37. Thecomposition according to claim 33, wherein said composition is effectiveto kill or inhibit the growth and/or proliferation of Trichophytonrubrum and said composition comprises one or more peptides, the aminoacid sequences of said peptides comprising one or more sequencesselected from the group consisting of PF-168, PF-278, PF-283, PF-307,PF-527, PF-531, PF-547, PF-672, PF-0019, PF-C021, PF-C252, PF-S028. 38.The composition according to claim 19, wherein said composition iseffective to kill or inhibit the growth and/or proliferation of abacterium and said composition comprises one or more peptides, the aminoacid sequences of said peptides comprising one or more sequencesselected from the group consisting of PF-006, PF-530, PF-531, PF-538,PF-C163, PF-C239, PF-C252, PF-C287, PF-006, PF-148, PF-283, PF-307,PF-322, PF-497, PF-499, PF-527, PF-531, PF-545, PF-547, PF-672, PF-S028,PF-522, PF-531, PF-538, PF-600, PF-606, PF-672, PF-C239, PF-C252,PF-006, PF-168, PF-209, PF-527, PF-545, PF-583, PF-606, PF-672, PF-C252,PF-S028, PF-S028, PF-C252, PF-531, PF-527, PF-S028, PF-C163, PF-C239,PF-C252, PF-C287, PF-278, PF-283, PF-527, PF-531, PF-583, PF-606,PF-672, PF-C163, PF-C252, PF-S028, PF-531, PF-547, PF-601, PF-0019,PF-C239, PF-C252, PF-S028, PF-437, PF-448, PF-511, PF-512, PF-520,PF-521, PF-523, PF-524, PF-525, PF-529, PF-600, and PF-606.
 39. Thecomposition according to claim 38, wherein said composition is effectiveto kill or inhibit the growth and/or proliferation of a gram positivebacterium and said composition comprises one or more peptides, the aminoacid sequences of said peptides comprising one or more sequencesselected from the group consisting of PF-006, PF-148, PF-168, PF-209,PF-278, PF-283, PF-307, PF-322, PF-437, PF-448, PF-497, PF-499, PF-511,PF-512, PF-520, PF-521, PF-522, PF-523, PF-524, PF-525, PF-527, PF-529,PF-531, PF-538, PF-545, PF-547, PF-583, PF-600, PF-601, PF-606, PF-672,PF-0019, PF-C163, PF-C239, PF-C252, PF-C287, PF-S028.
 40. Thecomposition according to claim 39, wherein said composition is effectiveto kill or inhibit the growth and/or proliferation of Actinomycesnaeslundii and said composition comprises one or more peptides, theamino acid sequences of said peptides comprising one or more sequencesselected from the group consisting of PF-C163, PF-C239, PF-C252, andPF-C287.
 41. The composition according to claim 39, wherein saidcomposition is effective to kill or inhibit the growth and/orproliferation of Bacillus subtilis and said composition comprises one ormore peptides, the amino acid sequences of said peptides comprising oneor more sequences selected from the group consisting of PF-006, PF-148,PF-283, PF-307, PF-322, PF-497, PF-499, PF-527, PF-531, PF-545, PF-547,PF-672, and PF-S028.
 42. The composition according to claim 39, whereinsaid composition is effective to kill or inhibit the growth and/orproliferation of Clostridium difficile and said composition comprisesone or more peptides, the amino acid sequences of said peptidescomprising one or more sequences selected from the group consisting ofPF-522, PF-531, and PF-538.
 43. The composition according to claim 39,wherein said composition is effective to kill or inhibit the growthand/or proliferation of Enterococcus faecalis and said compositioncomprises one or more peptides comprising the amino acid sequence ofPF-672.
 44. The composition according to claim 39, wherein saidcomposition is effective to kill or inhibit the growth and/orproliferation of Methicillin-resistant Staphylococcus aureus (MRSA) andsaid composition comprises one or more peptides, the amino acidsequences of said peptides comprising one or more sequences selectedfrom the group consisting of PF-006, PF-168, PF-209, PF-527, PF-545,PF-583, PF-606, PF-672, PF-C252, and PF-S028.
 45. The compositionaccording to claim 39, wherein said composition is effective to kill orinhibit the growth and/or proliferation of S. epidermidis and saidcomposition comprises one or more peptides, the amino acid sequences ofsaid peptides comprising one or more sequences selected from the groupconsisting of PF-278, PF-283, PF-527, PF-531, PF-583, PF-606, PF-672,PF-C163, PF-C252, and PF-S028.
 46. The composition according to claim39, wherein said composition is effective to kill or inhibit the growthand/or proliferation of Streptococcus mutans and said compositioncomprises one or more peptides, the amino acid sequences of saidpeptides comprising one or more sequences selected from the groupconsisting of PF-531, PF-547, PF-601, PF-0019, PF-C239, PF-C252, andPF-S028.
 47. The composition according to claim 39, wherein saidcomposition is effective to kill or inhibit the growth and/orproliferation of Streptococcus pneumoniae and said composition comprisesone or more peptides, the amino acid sequences of said peptidescomprising one or more sequences selected from the group consisting ofPF-437, PF-448, PF-511, PF-512, PF-520, PF-521, PF-523, PF-524, PF-525,PF-529, PF-600, and PF-606.
 48. The composition according to claim 39,wherein said composition is effective to kill or inhibit the growthand/or proliferation of Corynebacterium jeikium and said compositioncomprises one or more peptides, the amino acid sequences of saidpeptides comprising one or more sequences selected from the groupconsisting of PF-531, PF-S028, PF-527.
 49. The composition according toclaim 38, wherein said composition is effective to kill or inhibit thegrowth and/or proliferation of a gram negative bacterium and saidcomposition comprises one or more peptides, the amino acid sequences ofsaid peptides comprising one or more sequences selected from the groupconsisting of PF-006, PF-527, PF-530, PF-531, PF-538, PF-600, PF-606,PF-C163, PF-C239, PF-C252, PF-C287, and PF-S028.
 50. The compositionaccording to claim 49, wherein said composition is effective to kill orinhibit the growth and/or proliferation of Acinetobacter baumannii andsaid composition comprises one or more peptides, the amino acidsequences of said peptides comprising one or more sequences selectedfrom the group consisting of PF-006, PF-530, PF-531, and PF-538.
 51. Thecomposition according to claim 49, wherein said composition is effectiveto kill or inhibit the growth and/or proliferation of Escherichia coliand said composition comprises one or more peptides, the amino acidsequences of said peptides comprising one or more sequences selectedfrom the group consisting of PF-600, and PF-606.
 52. The compositionaccording to claim 49, wherein said composition is effective to kill orinhibit the growth and/or proliferation of Fusobacterium nucleatum andsaid composition comprises one or more peptides, the amino acidsequences of said peptides comprising one or more sequences selectedfrom the group consisting of PF-C239, and PF-C252.
 53. The compositionaccording to claim 49, wherein said composition is effective to kill orinhibit the growth and/or proliferation of Pseudomonas aeruginosa andsaid composition comprises one or more peptides, the amino acidsequences of said peptides comprising one or more sequences selectedfrom the group consisting of PF-S028, PF-C252, PF-531, and PF-527. 54.The composition according to claim 49, wherein said composition iseffective to kill or inhibit the growth and/or proliferation ofPorphyromonas gingivalis and said composition comprises one or morepeptides, the amino acid sequences of said peptides comprising one ormore sequences selected from the group consisting of PF-S028, PF-C163,PF-C239, PF-C252, and PF-C287. 55-57. (canceled)
 58. The composition ofclaim 33, wherein said peptide comprises one or more protecting groups.59-63. (canceled)
 64. A method of killing and/or inhibiting the growthand/or proliferation of a microorganism, said method comprisingcontacting said microorganism with a peptide of claim 1, a construct ofclaim 20, or a composition of claim
 32. 65-69. (canceled)
 70. A methodof disinfecting a surface, said method comprising contacting saidsurface with one or more peptides of claim 1 or a composition of claim32.
 71. The method of claim 70, wherein said surface comprises a surfaceof a prosthesis or medical implant.
 72. The method of claim 70, whereinsaid surface comprises a surface of a medical device.
 73. The method ofclaim 70, wherein said surface comprises a surface of a plant orfoodstuff.
 74. The method of claim 70, wherein said peptide(s) arecombined with a second disinfectant selected from the group consistingof other antimicrobial agent is a disinfectant selected from the groupconsisting of acetic acid, phosphoric acid, citric acid, lactic, formic,propionic acid, hydrochloric acid, sulfuric acid, nitric acid, sodiumhydroxide, potassium hydroxide, sodium carbonate, ammonium hydroxide,ethyl alcohol, isopropyl alcohol, phenol, formaldehyde, glutaraldehyde,hypochlorites, chlorine dioxide, sodium dichloroisocyanurate,chloramine-T, iodine, povidone-iodine, chlorhexidine, hydrogen peroxide,peracetic acid, and benzalkonium chloride.